Non-viral dna vectors and uses thereof for expressing fviii therapeutics

ABSTRACT

The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding FVIII protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of FVIII protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of FVIII protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing FVIII protein. Such FVIII protein can be expressed for treating disease, e.g., hemophilia A.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/817,904, filed on Mar. 13, 2019 and U.S. Provisional Application No.62/856,432, filed on Jun. 3, 2019, the contents of each of which arehereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format, as well as sequences in Tables1-9 herein, and each are hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 11, 2020, is named131698-06020_SL.txt and is 116,781 bytes in size

TECHNICAL FIELD

The present invention relates to the field of gene therapy, includingnon-viral vectors for expressing a transgene or isolated polynucleotidesin a subject or cell. The disclosure also relates to nucleic acidconstructs, promoters, vectors, and host cells including thepolynucleotides as well as methods of delivering exogenous DNA sequencesto a target cell, tissue, organ or organism. For example, the presentdisclosure provides methods for using non-viral ceDNA vectors to expressFVIII, from a cell, e.g., expressing the FVIII therapeutic protein forthe treatment of a subject with a hemophilia A. The methods andcompositions can be used e.g., for treating disease by expressing theFVIII in a cell or tissue of a subject in need thereof.

BACKGROUND

Gene therapy aims to improve clinical outcomes for patients sufferingfrom either genetic mutations or acquired diseases caused by anaberration in the gene expression profile. Gene therapy includes thetreatment or prevention of medical conditions resulting from defectivegenes or abnormal regulation or expression, e.g. underexpression oroverexpression, that can result in a disorder, disease, malignancy, etc.For example, a disease or disorder caused by a defective gene might betreated, prevented or ameliorated by delivery of a corrective geneticmaterial to a patient, or might be treated, prevented or ameliorated byaltering or silencing a defective gene, e.g., with a corrective geneticmaterial to a patient resulting in the therapeutic expression of thegenetic material within the patient.

The basis of gene therapy is to supply a transcription cassette with anactive gene product (sometimes referred to as a transgene), e.g., thatcan result in a positive gain-of-function effect, a negativeloss-of-function effect, or another outcome. Such outcomes can beattributed to expression of a therapeutic protein such as an antibody, afunctional enzyme, or a fusion protein. Gene therapy can also be used totreat a disease or malignancy caused by other factors. Human monogenicdisorders can be treated by the delivery and expression of a normal geneto the target cells. Delivery and expression of a corrective gene in thepatient's target cells can be carried out via numerous methods,including the use of engineered viruses and viral gene delivery vectors.Among the many virus-derived vectors available (e.g., recombinantretrovirus, recombinant lentivirus, recombinant adenovirus, and thelike), recombinant adeno-associated virus (rAAV) is gaining popularityas a versatile vector in gene therapy.

Adeno-associated viruses (AAV) belong to the Parvoviridae family andmore specifically constitute the dependoparvovirus genus. Vectorsderived from AAV (i.e., recombinant AAV (rAVV) or AAV vectors) areattractive for delivering genetic material because (i) they are able toinfect (transduce) a wide variety of non-dividing and dividing celltypes including myocytes and neurons; (ii) they are devoid of the virusstructural genes, thereby diminishing the host cell responses to virusinfection, e.g., interferon-mediated responses; (iii) wild-type virusesare considered non-pathologic in humans; (iv) in contrast to wild typeAAV, which are capable of integrating into the host cell genome,replication-deficient AAV vectors lack the rep gene and generallypersist as episomes, thus limiting the risk of insertional mutagenesisor genotoxicity; and (v) in comparison to other vector systems, AAVvectors are generally considered to be relatively poor immunogens andtherefore do not trigger a significant immune response (see ii), thusgaining persistence of the vector DNA and potentially, long-termexpression of the therapeutic transgenes.

However, there are several major deficiencies in using AAV particles asa gene delivery vector. One major drawback associated with rAAV is itslimited viral packaging capacity of about 4.5 kb of heterologous DNA(Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), andas a result, use of AAV vectors has been limited to less than 150,000 Daprotein coding capacity. The second drawback is that as a result of theprevalence of wild-type AAV infection in the population, candidates forrAAV gene therapy have to be screened for the presence of neutralizingantibodies that eliminate the vector from the patient. A third drawbackis related to the capsid immunogenicity that prevents re-administrationto patients that were not excluded from an initial treatment. The immunesystem in the patient can respond to the vector which effectively actsas a “booster” shot to stimulate the immune system generating high titeranti-AAV antibodies that preclude future treatments. Some recent reportsindicate concerns with immunogenicity in high dose situations. Anothernotable drawback is that the onset of AAV-mediated gene expression isrelatively slow, given that single-stranded AAV DNA must be converted todouble-stranded DNA prior to heterologous gene expression.

Additionally, conventional AAV virions with capsids are produced byintroducing a plasmid or plasmids containing the AAV genome, rep genes,and cap genes (Grimm et al., 1998). However, such encapsidated AAV virusvectors were found to inefficiently transduce certain cell and tissuetypes and the capsids also induce an immune response.

Accordingly, use of adeno-associated virus (AAV) vectors for genetherapy is limited due to the single administration to patients (owingto the patient immune response), the limited range of transgene geneticmaterial suitable for delivery in AAV vectors due to minimal viralpackaging capacity (about 4.5 kb), and slow AAV-mediated geneexpression.

There is large unmet need for disease-modifying therapies in hemophiliaA. Current therapies are burdensome and require intravenous (IV)administrations. First, these Factor VIII injectables do not providecontinuous delivery of factors, with trough levels allowing bleedingepisodes. Second, there are no approved gene therapies for hemophilia A,and AAV based therapies cannot be used by 25% to 40% of patients due topre-existing antibodies. AAV can only be administered once, and theresulting Factor VIII levels might not be high enough to be efficacious,or may be supranormal, dose levels cannot be titrated. Third, manyhemophilia A patients cannot utilize these therapies because of thedevelopment of neutralizing antibodies to these exogenous, artificialclotting factors.

Accordingly, there is need in the field for a technology that permitsexpression of a therapeutic FVIII protein in a cell, tissue or subjectfor the treatment of hemophilia A.

BRIEF DESCRIPTION

The technology described herein relates to methods and compositions fortreatment of Hemophilia A by expression of Factor VIII (FVIII) proteinfrom a capsid-free (e.g., non-viral) DNA vector with covalently-closedends (referred to herein as a “closed-ended DNA vector” or a “ceDNAvector”), where the ceDNA vector comprises a FVIII nucleic acid sequenceor codon optimized versions thereof. These ceDNA vector can be used toproduce FVIII proteins for treatment, monitoring, and diagnosis. Theapplication of ceDNA vectors expressing FVIII to the subject for thetreatment of hemophilia A is useful to: (i) provide disease modifyinglevels of FVIII enzyme, (ii) be minimally invasive in delivery, (iii) berepeatable and dosed-to-effect, (iv) have rapid onset of therapeuticeffect, (v) result in sustained expression of corrective FVIII enzyme inthe liver, (vi) restore urea cycle function, and/or (vii) be titratableto achieve the appropriate pharmacologic levels of the defective enzyme.

In some embodiments, a ceDNA-vector expressing FVIII is optionallypresent in a liposome nanoparticle formulation (LNP) to the treatment ofhemophilia A. The ceDNA vectors described herein can provide one or morebenefits including, but not limited to providing disease modifyinglevels of Factor VIII, being minimally invasive in delivery, beingrepeatable and dosed-to-effect, providing a rapid onset of therapeuticeffect, within days of therapeutic intervention, sustained expression ofcorrective Factor VIII levels in the circulation, be titratable toachieve the appropriate pharmacologic levels of the defectivecoagulation factor, and/or provide treatments for other hemophilia,including but not limited to Factor VIII deficiency.

Accordingly, the invention described herein relates to a capsid-free(e.g., non-viral) DNA vector with covalently-closed ends (referred toherein as a “closed-ended DNA vector” or a “ceDNA vector”) comprising aheterogeneous gene encoding FVIII, to permit expression of the FVIIItherapeutic protein in a cell. According to one aspect, the disclosureprovides a capsid-free close-ended DNA (ceDNA) vector comprising atleast one heterologous nucleotide sequence between flanking invertedterminal repeats (ITRs), wherein at least one heterologous nucleotidesequence encodes at least one FVIII protein, wherein the least oneheterologous nucleotide sequence that encodes at least one FVIII proteinis selected from any of the sequences in Table 1. According to someembodiments, the ceDNA vector comprises a nucleic acid sequence selectedfrom any of those in Table 9. According to some embodiments, the ceDNAvector comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:199, SEQ ID NO: 208, SEQ ID NO: 211 and SEQ ID NO: 214. According tosome embodiments, the ceDNA vector comprises a nucleic acid sequencethat is at least 85% identical to SEQ ID NO: 210. According to someembodiments, the ceDNA vector comprises a nucleic acid sequence that isat least 90% identical to SEQ ID NO: 210. According to some embodiments,the ceDNA vector comprises a nucleic acid sequence that is at least 95%identical to SEQ ID NO: 210. According to some embodiments, the ceDNAvector comprises a nucleic acid sequence that is at least 96% identicalto SEQ ID NO: 210. According to some embodiments, the ceDNA vectorcomprises a nucleic acid sequence that is at least 97% identical to SEQID NO: 210. According to some embodiments, the ceDNA vector comprises anucleic acid sequence that is at least 98% identical to SEQ ID NO: 210.According to some embodiments, the ceDNA vector comprises a nucleic acidsequence that is at least 99% identical to SEQ ID NO: 210. According tosome embodiments, the ceDNA vector comprises a nucleic acid sequencethat is at least 85% identical to SEQ ID NO: 214. According to someembodiments, the ceDNA vector comprises a nucleic acid sequence that isat least 90% identical to SEQ ID NO: 214. According to some embodiments,the ceDNA vector comprises a nucleic acid sequence that is at least 95%identical to SEQ ID NO: 214. According to some embodiments, the ceDNAvector comprises a nucleic acid sequence that is at least 96% identicalto SEQ ID NO: 214. According to some embodiments, the ceDNA vectorcomprises a nucleic acid sequence that is at least 97% identical to SEQID NO: 214. According to some embodiments, the ceDNA vector comprises anucleic acid sequence that is at least 98% identical to SEQ ID NO: 214.According to some embodiments, the ceDNA vector comprises a nucleic acidsequence that is at least 99% identical to SEQ ID NO: 214.

In one embodiment, disclosed herein is a capsid-free close-ended DNA(ceDNA) vector comprising at least one heterologous nucleotide sequencebetween flanking inverted terminal repeats (ITRs), wherein at least oneheterologous nucleotide sequence encodes at least one FVIII protein,wherein the least one heterologous nucleotide sequence that encodes atleast one FVIII protein is selected from a sequence having at least 85%identity to any sequence in Table 1. In one embodiment, the least oneheterologous nucleotide sequence that encodes at least one FVIII proteinis selected from a sequence having at least 90% identity to any sequencein Table 1. In one embodiment, the least one heterologous nucleotidesequence that encodes at least one FVIII protein is selected from asequence having at least 91% identity to any sequence in Table 1. In oneembodiment, the least one heterologous nucleotide sequence that encodesat least one FVIII protein is selected from a sequence having at least92% identity to any sequence in Table 1. In one embodiment, the leastone heterologous nucleotide sequence that encodes at least one FVIIIprotein is selected from a sequence having at least 93% identity to anysequence in Table 1. In one embodiment, the least one heterologousnucleotide sequence that encodes at least one FVIII protein is selectedfrom a sequence having at least 94% identity to any sequence in Table 1.In one embodiment, the least one heterologous nucleotide sequence thatencodes at least one FVIII protein is selected from a sequence having atleast 95% identity to any sequence in Table 1. In one embodiment, theleast one heterologous nucleotide sequence that encodes at least oneFVIII protein is selected from a sequence having at least 96% identityto any sequence in Table 1. In one embodiment, the least oneheterologous nucleotide sequence that encodes at least one FVIII proteinis selected from a sequence having at least 97% identity to any sequencein Table 1. In one embodiment, the least one heterologous nucleotidesequence that encodes at least one FVIII protein is selected from asequence having at least 98% identity to any sequence in Table 1. In oneembodiment, the least one heterologous nucleotide sequence that encodesat least one FVIII protein is selected from a sequence having at least99% identity to any sequence in Table 1. In one embodiment, the leastone heterologous nucleotide sequence that encodes at least one FVIIIprotein is selected from a sequence comprising any sequence in Table 1.In one embodiment, the least one heterologous nucleotide sequence thatencodes at least one FVIII protein is selected from a sequenceconsisting of any sequence in Table 1.

In one embodiment, the sequence in Table 1 is selected from the groupconsisting of SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ IDNO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392,SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, and SEQ IDNO:397. In one embodiment, the sequence is SEQ ID NO: 380. In oneembodiment, the sequence is SEQ ID NO:381. In one embodiment, thesequence is SEQ ID NO:382. In one embodiment, the sequence is SEQ IDNO:383. In one embodiment, the sequence is SEQ ID NO:384. In oneembodiment, the sequence is SEQ ID NO:385. In one embodiment, thesequence is SEQ ID NO:386. In one embodiment, the sequence is SEQ IDNO:387. In one embodiment, the sequence is SEQ ID NO:388. In oneembodiment, the sequence is SEQ ID NO:389. In one embodiment, thesequence is SEQ ID NO:390. In one embodiment, the sequence is SEQ IDNO:391. In one embodiment, the sequence is SEQ ID NO:392. In oneembodiment, the sequence is SEQ ID NO:393. In one embodiment, thesequence is SEQ ID NO:394. In one embodiment, the sequence is SEQ IDNO:395. In one embodiment, the sequence is SEQ ID NO:396. In oneembodiment, the sequence is SEQ ID NO:397.

In one embodiment, the ceDNA vector comprises a nucleic acid sequenceselected from a sequence having at least 85% identity with any sequencein Table 9. In one embodiment, the ceDNA vector comprises a nucleic acidsequence selected from a sequence having at least 90% identity with anysequence in Table 9. In one embodiment, the ceDNA vector comprises anucleic acid sequence selected from a sequence having at least 91%identity with any sequence in Table 9. In one embodiment, the ceDNAvector comprises a nucleic acid sequence selected from a sequence havingat least 92% identity with any sequence in Table 9. In one embodiment,the ceDNA vector comprises a nucleic acid sequence selected from asequence having at least 93% identity with any sequence in Table 9. Inone embodiment, the ceDNA vector comprises a nucleic acid sequenceselected from a sequence having at least 94% identity with any sequencein Table 9. In one embodiment, the ceDNA vector comprises a nucleic acidsequence selected from a sequence having at least 95% identity with anysequence in Table 9. In one embodiment, the ceDNA vector comprises anucleic acid sequence selected from a sequence having at least 96%identity with any sequence in Table 9. In one embodiment, the ceDNAvector comprises a nucleic acid sequence selected from a sequence havingat least 97% identity with any sequence in Table 9. In one embodiment,the ceDNA vector comprises a nucleic acid sequence selected from asequence having at least 98% identity with any sequence in Table 9. Inone embodiment, the ceDNA vector comprises a nucleic acid sequenceselected from a sequence having at least 99% identity with any sequencein Table 9. In one embodiment, the ceDNA vector comprises a nucleic acidsequence comprising a sequence in Table 9. In one embodiment, the ceDNAvector comprises a nucleic acid sequence consisting of a sequence inTable 9.

In one embodiment, the sequence in Table 9 is selected from the groupconsisting of SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ IDNO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209,SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ IDNO:214, SEQ ID NO:215, SEQ ID NO:216, and SEQ ID NO:217. In oneembodiment, the sequence in Table 9 is SEQ ID NO:197. In one embodiment,the sequence in Table 9 is SEQ ID NO:198. In one embodiment, thesequence in Table 9 is SEQ ID NO:199. In one embodiment, the sequence inTable 9 is 200. In one embodiment, the sequence in Table 9 is 201. Inone embodiment, the sequence in Table 9 is 202. In one embodiment, thesequence in Table 9 is 203. In one embodiment, the sequence in Table 9is 204. In one embodiment, the sequence in Table 9 is 205. In oneembodiment, the sequence in Table 9 is 206. In one embodiment, thesequence in Table 9 is 207. In one embodiment, the sequence in Table 9is 208. In one embodiment, the sequence in Table 9 is 209. In oneembodiment, the sequence in Table 9 is 210. In one embodiment, thesequence in Table 9 is 211. In one embodiment, the sequence in Table 9is 212. In one embodiment, the sequence in Table 9 is 213. In oneembodiment, the sequence in Table 9 is 214. In one embodiment, thesequence in Table 9 is 215. In one embodiment, the sequence in Table 9is 216. In one embodiment, the sequence in Table 9 is 217.

In one embodiment, the ceDNA vector comprises anucleic acid sequencehaving at least 85% identity to SEQ ID NO:210. In one embodiment, theceDNA vector comprises anucleic acid sequence having at least 90%identity to SEQ ID NO:210. In one embodiment, the ceDNA vector comprisesanucleic acid sequence having at least 91% identity to SEQ ID NO:210. Inone embodiment, the ceDNA vector comprises anucleic acid sequence havingat least 92% identity to SEQ ID NO:210. In one embodiment, the ceDNAvector comprises anucleic acid sequence having at least 93% identity toSEQ ID NO:210. In one embodiment, the ceDNA vector comprises anucleicacid sequence having at least 94% identity to SEQ ID NO:210. In oneembodiment, the ceDNA vector comprises anucleic acid sequence having atleast 95% identity to SEQ ID NO:210. In one embodiment, the ceDNA vectorcomprises anucleic acid sequence having at least 96% identity to SEQ IDNO:210. In one embodiment, the ceDNA vector comprises anucleic acidsequence having at least 97% identity to SEQ ID NO:210. In oneembodiment, the ceDNA vector comprises anucleic acid sequence having atleast 98% identity to SEQ ID NO:210. In one embodiment, the ceDNA vectorcomprises anucleic acid sequence having at least 99% identity to SEQ IDNO:210. In one embodiment, the ceDNA vector comprises anucleic acidsequence comprising SEQ ID NO:210. In one embodiment, the ceDNA vectorcomprises anucleic acid sequence consisting of SEQ ID NO:210.

In one embodiment, the ceDNA vector comprises anucleic acid sequencehaving at least 85% identity to SEQ ID NO:214. In one embodiment, theceDNA vector comprises anucleic acid sequence having at least 90%identity to SEQ ID NO:214. In one embodiment, the ceDNA vector comprisesanucleic acid sequence having at least 91% identity to SEQ ID NO:214. Inone embodiment, the ceDNA vector comprises anucleic acid sequence havingat least 92% identity to SEQ ID NO:214. In one embodiment, the ceDNAvector comprises anucleic acid sequence having at least 93% identity toSEQ ID NO:214. In one embodiment, the ceDNA vector comprises anucleicacid sequence having at least 94% identity to SEQ ID NO:214. In oneembodiment, the ceDNA vector comprises anucleic acid sequence having atleast 95% identity to SEQ ID NO:214. In one embodiment, the ceDNA vectorcomprises anucleic acid sequence having at least 96% identity to SEQ IDNO:214. In one embodiment, the ceDNA vector comprises anucleic acidsequence having at least 97% identity to SEQ ID NO:214. In oneembodiment, the ceDNA vector comprises anucleic acid sequence having atleast 98% identity to SEQ ID NO:214. In one embodiment, the ceDNA vectorcomprises anucleic acid sequence having at least 99% identity to SEQ IDNO:214. In one embodiment, the ceDNA vector comprises anucleic acidsequence comprising SEQ ID NO:214. In one embodiment, the ceDNA vectorcomprises anucleic acid sequence consisting of SEQ ID NO:214.

The ceDNA vectors for expression of FVIII protein production asdescribed herein are capsid-free, linear duplex DNA molecules formedfrom a continuous strand of complementary DNA with covalently-closedends (linear, continuous and non-encapsidated structure), which comprisea 5′ inverted terminal repeat (ITR) sequence and a 3′ ITR sequence,where the 5′ ITR and the 3′ ITR can have the same symmetricalthree-dimensional organization with respect to each other, (i.e.,symmetrical or substantially symmetrical), or alternatively, the 5′ ITRand the 3′ ITR can have different three-dimensional organization withrespect to each other (i.e., asymmetrical ITRs). In addition, the ITRscan be from the same or different serotypes. In some embodiments, aceDNA vector can comprise ITR sequences that have a symmetricalthree-dimensional spatial organization such that their structure is thesame shape in geometrical space, or have the same A, C-C′ and B-B′ loopsin 3D space (i.e., they are the same or are mirror images with respectto each other). In some embodiments, one ITR can be from one AAVserotype, and the other ITR can be from a different AAV serotype.

Accordingly, some aspects of the technology described herein relate to aceDNA vector for improved protein expression and/or production of theabove described FVIII protein that comprise ITR sequences that flank aheterologous nucleic acid sequence comprising any FVIII nucleic acidsequence disclosed in Tables 5, the ITR sequences being selected fromany of: (i) at least one WT ITR and at least one modified AAV invertedterminal repeat (ITR) (e.g., asymmetric modified ITRs); (ii) twomodified ITRs where the mod-ITR pair have a different three-dimensionalspatial organization with respect to each other (e.g., asymmetricmodified ITRs), or (iii) symmetrical or substantially symmetrical WT-WTITR pair, where each WT-ITR has the same three-dimensional spatialorganization, or (iv) symmetrical or substantially symmetrical modifiedITR pair, where each mod-ITR has the same three-dimensional spatialorganization. The ceDNA vectors disclosed herein can be produced ineukaryotic cells, thus devoid of prokaryotic DNA modifications andbacterial endotoxin contamination in insect cells.

The methods and compositions described herein relate, in part, to thediscovery of a non-viral capsid-free DNA vector with covalently-closedends (ceDNA vectors) that can be used to express at least one FVIIIprotein, or more than one FVIII protein from a cell, including but notlimited to cells of the liver.

Accordingly, provided herein in one aspect are DNA vectors (e.g., ceDNAvectors) comprising at least one heterologous nucleic acid sequenceencoding at least one transgene encoding FVIII proteins thereof operablylinked to a promoter positioned between two different AAV invertedterminal repeat sequences (ITRs), one of the ITRS comprising afunctional AAV terminal resolution site and a Rep binding site, and oneof the ITRs comprising a deletion, insertion, or substitution relativeto the other ITR; wherein the transgene encodes an FVIII protein; andwherein the DNA when digested with a restriction enzyme having a singlerecognition site on the DNA vector has the presence of characteristicbands of linear and continuous DNA as compared to linear andnon-continuous DNA controls when analyzed on a non-denaturing gel. Otheraspects include delivery of the FVIII protein by expressing it in vivofrom a ceDNA vector as described herein and further, the treatment ofhemophilia A using ceDNA vectors encoding the FVIII. Also contemplatedherein are cells comprising a ceDNA vector encoding FVIII as describedherein. According to some embodiments, the ceDNA vector is selected fromthe group consisting of ceDNAFVIII-vector 16, ceDNAFVIII-vector 19,ceDNAFVIII-vector 21, ceDNAFVIII-vector 8, ceDNAFVIII-vector 17,ceDNAFVIII-vector 20 and ceDNAFVIII-vector 23. According to someembodiments, the ceDNA vector is ceDNAFVIII vector 19 or ceDNAFVIIIvector 23.

Aspects of the invention relate to methods to produce the ceDNA vectorsuseful for FVIII protein expression in a cell as described herein. Otherembodiments relate to a ceDNA vector produced by the method providedherein. In one embodiment, the capsid free (e.g., non-viral) DNA vector(ceDNA vector) for FVIII protein production is obtained from a plasmid(referred to herein as a “ceDNA-plasmid”) comprising a polynucleotideexpression construct template comprising in this order: a first 5′inverted terminal repeat (e.g. AAV ITR); a heterologous nucleic acidsequence; and a 3′ ITR (e.g. AAV ITR), where the 5′ ITR and 3′ITR can beasymmetric relative to each other, or symmetric (e.g., WT-ITRs ormodified symmetric ITRs) as defined herein. According to someembodiments, the ceDNA vector is selected from the group consisting ofceDNAFVIII-vector 16, ceDNAFVIII-vector 19, ceDNAFVIII-vector 21,ceDNAFVIII-vector 8, ceDNAFVIII-vector 17, ceDNAFVIII-vector 20 andceDNAFVIII-vector 23. According to some embodiments, the ceDNA vector isceDNAFVIII vector 19 or ceDNAFVIII vector 23.

The ceDNA vector for expression of the FVIII protein as disclosed hereinis obtainable by a number of means that would be known to the ordinarilyskilled artisan after reading this disclosure. For example, apolynucleotide expression construct template used for generating theceDNA vectors of the present invention can be a ceDNA-plasmid, aceDNA-bacmid, and/or a ceDNA-baculovirus. In one embodiment, theceDNA-plasmid comprises a restriction cloning site (e.g. SEQ ID NO: 123and/or 124) operably positioned between the ITRs where an expressioncassette comprising e.g., a promoter operatively linked to a transgene,e.g., a nucleic acid encoding FVIII can be inserted. In someembodiments, ceDNA vectors for expression of FVIII protein are producedfrom a polynucleotide template (e.g., ceDNA-plasmid, ceDNA-bacmid,ceDNA-baculovirus) containing symmetric or asymmetric ITRs (modified orWT ITRs).

In a permissive host cell, in the presence of e.g., Rep, thepolynucleotide template having at least two ITRs replicates to produceceDNA vectors expressing the FVIII protein. ceDNA vector productionundergoes two steps: first, excision (“rescue”) of template from thetemplate backbone (e.g. ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirusgenome etc.) via Rep proteins, and second, Rep mediated replication ofthe excised ceDNA vector. Rep proteins and Rep binding sites of thevarious AAV serotypes are well known to those of ordinary skill in theart. One of ordinary skill understands to choose a Rep protein from aserotype that binds to and replicates the nucleic acid sequence basedupon at least one functional ITR. For example, if the replicationcompetent ITR is from AAV serotype 2, the corresponding Rep would befrom an AAV serotype that works with that serotype such as AAV2 ITR withAAV2 or AAV4 Rep but not AAV5 Rep, which does not. Upon replication, thecovalently-closed ended ceDNA vector continues to accumulate inpermissive cells and ceDNA vector is preferably sufficiently stable overtime in the presence of Rep protein under standard replicationconditions, e.g. to accumulate in an amount that is at least 1 pg/cell,preferably at least 2 pg/cell, preferably at least 3 pg/cell, morepreferably at least 4 pg/cell, even more preferably at least 5 pg/cell.

Accordingly, one aspect of the invention relates to a process ofproducing a ceDNA vector for expression of such FVIII proteinscomprising the steps of: a) incubating a population of host cells (e.g.insect cells) harboring the polynucleotide expression construct template(e.g., a ceDNA-plasmid, a ceDNA-bacmid, and/or a ceDNA-baculovirus),which is devoid of viral capsid coding sequences, in the presence of aRep protein under conditions effective and for a time sufficient toinduce production of the ceDNA vector within the host cells, and whereinthe host cells do not comprise viral capsid coding sequences; and b)harvesting and isolating the ceDNA vector from the host cells. Thepresence of Rep protein induces replication of the vector polynucleotidewith a modified ITR to produce the ceDNA vector for expression of FVIIIprotein in a host cell. However, no viral particles (e.g. AAV virions)are expressed. Thus, there is no virion-enforced size limitation.According to some embodiments, the ceDNA vector is selected from thegroup consisting of ceDNAFVIII-vector 16, ceDNAFVIII-vector 19,ceDNAFVIII-vector 21, ceDNAFVIII-vector 8, ceDNAFVIII-vector 17,ceDNAFVIII-vector 20 and ceDNAFVIII-vector 23. According to someembodiments, the ceDNA vector is ceDNAFVIII vector 19 or ceDNAFVIIIvector 23.

The presence of the ceDNA vector useful for expression of FVIII proteinis isolated from the host cells can be confirmed by digesting DNAisolated from the host cell with a restriction enzyme having a singlerecognition site on the ceDNA vector and analyzing the digested DNAmaterial on denaturing and non-denaturing gels to confirm the presenceof characteristic bands of linear and continuous DNA as compared tolinear and non-continuous DNA.

Also provided herein are methods of expressing an FVIII protein that hastherapeutic uses, using a ceDNA vector in a cell or subject. Such FVIIIproteins can be used for the treatment of hemophilia A. Accordingly,provided herein are methods for the treatment of hemophilia A comprisingadministering a ceDNA vector encoding a therapeutic FVIII protein to asubject in need thereof. According to some embodiments, the ceDNA vectorencoding a therapeutic FVIII protein is selected from the groupconsisting of ceDNAFVIII-vector 16, ceDNAFVIII-vector 19,ceDNAFVIII-vector 21, ceDNAFVIII-vector 8, ceDNAFVIII-vector 17,ceDNAFVIII-vector 20 and ceDNAFVIII-vector 23. According to someembodiments, the ceDNA vector is ceDNAFVIII vector 19 or ceDNAFVIIIvector 23. According to some embodiments, levels of FVIII in the serumof the subject are increased in subjects administered the ceDNA vectorcompared to a control. According to some embodiments, the increase inlevels of FVIII is greater than about 40% compared to the control.According to some embodiments, the increase in levels of FVIII isgreater than about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75% compared to the control.

In some embodiments, one aspect of the technology described hereinrelates to a non-viral capsid-free DNA vector with covalently-closedends (ceDNA vector), wherein the ceDNA vector comprises at least oneheterologous nucleotide sequence, operably positioned between twoinverted terminal repeat sequences where the ITR sequences can beasymmetric, or symmetric, or substantially symmetrical as these termsare defined herein, wherein at least one of the ITRs comprises afunctional terminal resolution site and a Rep binding site, andoptionally the heterologous nucleic acid sequence encodes a transgene(e.g., FVIII protein) and wherein the vector is not in a viral capsid.

These and other aspects of the invention are described in further detailbelow.

DESCRIPTION OF DRAWINGS

Embodiments of the present disclosure, briefly summarized above anddiscussed in greater detail below, can be understood by reference to theillustrative embodiments of the disclosure depicted in the appendeddrawings. However, the appended drawings illustrate only typicalembodiments of the disclosure and are therefore not to be consideredlimiting of scope, for the disclosure may admit to other equallyeffective embodiments.

FIG. 1A illustrates an exemplary structure of a ceDNA vector forexpression of an FVIII protein as disclosed herein, comprisingasymmetric ITRs. In this embodiment, the exemplary ceDNA vectorcomprises an expression cassette containing CAG promoter, WPRE, andBGHpA. An open reading frame (ORF) encoding the FVIII transgene can beinserted into the cloning site (R3/R4) between the CAG promoter andWPRE. The expression cassette is flanked by two inverted terminalrepeats (ITRs)—the wild-type AAV2 ITR on the upstream (5′-end) and themodified ITR on the downstream (3′-end) of the expression cassette,therefore the two ITRs flanking the expression cassette are asymmetricwith respect to each other.

FIG. 1B illustrates an exemplary structure of a ceDNA vector forexpression of the FVIII as disclosed herein comprising asymmetric ITRswith an expression cassette containing CAG promoter, WPRE, and BGHpA. Anopen reading frame (ORF) encoding the FVIII transgene can be insertedinto the cloning site between CAG promoter and WPRE. The expressioncassette is flanked by two inverted terminal repeats (ITRs)—a modifiedITR on the upstream (5′-end) and a wild-type ITR on the downstream(3′-end) of the expression cassette.

FIG. 1C illustrates an exemplary structure of a ceDNA vector forexpression of the FVIII as disclosed herein comprising asymmetric ITRs,with an expression cassette containing an enhancer/promoter, the FVIIItransgene, a post transcriptional element (WPRE), and a polyA signal. Anopen reading frame (ORF) allows insertion of the FVIII transgene intothe cloning site between CAG promoter and WPRE. The expression cassetteis flanked by two inverted terminal repeats (ITRs) that are asymmetricalwith respect to each other; a modified ITR on the upstream (5′-end) anda modified ITR on the downstream (3′-end) of the expression cassette,where the 5′ ITR and the 3′ITR are both modified ITRs but have differentmodifications (i.e., they do not have the same modifications).

FIG. 1D illustrates an exemplary structure of a ceDNA vector forexpression of the FVIII as disclosed herein, comprising symmetricmodified ITRs, or substantially symmetrical modified ITRs as definedherein, with an expression cassette containing CAG promoter, WPRE, andBGHpA. An open reading frame (ORF) encoding the FVIII transgene isinserted into the cloning site between CAG promoter and WPRE. Theexpression cassette is flanked by two modified inverted terminal repeats(ITRs), where the 5′ modified ITR and the 3′ modified ITR aresymmetrical or substantially symmetrical.

FIG. 1E illustrates an exemplary structure of a ceDNA vector forexpression of the FVIII as disclosed herein comprising symmetricmodified ITRs, or substantially symmetrical modified ITRs as definedherein, with an expression cassette containing an enhancer/promoter, atransgene, a post transcriptional element (WPRE), and a polyA signal. Anopen reading frame (ORF) allows insertion of a transgene (e.g., theFVIII) into the cloning site between CAG promoter and WPRE. Theexpression cassette is flanked by two modified inverted terminal repeats(ITRs), where the 5′ modified ITR and the 3′ modified ITR aresymmetrical or substantially symmetrical.

FIG. 1F illustrates an exemplary structure of a ceDNA vector forexpression of the FVIII as disclosed herein, comprising symmetricWT-ITRs, or substantially symmetrical WT-ITRs as defined herein, with anexpression cassette containing CAG promoter, WPRE, and BGHpA. An openreading frame (ORF) encoding a transgene (e.g., the FVIII) is insertedinto the cloning site between CAG promoter and WPRE. The expressioncassette is flanked by two wild type inverted terminal repeats(WT-ITRs), where the 5′ WT-ITR and the 3′ WT ITR are symmetrical orsubstantially symmetrical.

FIG. 1G illustrates an exemplary structure of a ceDNA vector forexpression of the FVIII as disclosed herein, comprising symmetricmodified ITRs, or substantially symmetrical modified ITRs as definedherein, with an expression cassette containing an enhancer/promoter, atransgene (e.g., the FVIII), a post transcriptional element (WPRE), anda polyA signal. An open reading frame (ORF) allows insertion of atransgene (e.g., the FVIII) into the cloning site between CAG promoterand WPRE. The expression cassette is flanked by two wild type invertedterminal repeats (WT-ITRs), where the 5′ WT-ITR and the 3′ WT ITR aresymmetrical or substantially symmetrical.

FIG. 2A provides the T-shaped stem-loop structure of a wild-type leftITR of AAV2 (SEQ ID NO: 52) with identification of A-A′ arm, B-B′ arm,C-C′ arm, two Rep binding sites (RBE and RBE′) and also shows theterminal resolution site (TRS). The RBE contains a series of 4 duplextetramers that are believed to interact with either Rep 78 or Rep 68. Inaddition, the RBE′ is also believed to interact with Rep complexassembled on the wild-type ITR or mutated ITR in the construct. The Dand D′ regions contain transcription factor binding sites and otherconserved structure. FIG. 2B shows proposed Rep-catalyzed nicking andligating activities in a wild-type left ITR (SEQ ID NO: 53), includingthe T-shaped stem-loop structure of the wild-type left ITR of AAV2 withidentification of A-A′ arm, B-B′ arm, C-C′ arm, two Rep Binding sites(RBE and RBE′) and also shows the terminal resolution site (TRS), andthe D and D′ region comprising several transcription factor bindingsites and other conserved structure.

FIG. 3A provides the primary structure (polynucleotide sequence) (left)and the secondary structure (right) of the RBE-containing portions ofthe A-A′ arm, and the C-C′ and B-B′ arm of the wild type left AAV2 ITR(SEQ ID NO: 54). FIG. 3B shows an exemplary mutated ITR (also referredto as a modified ITR) sequence for the left ITR. Shown is the primarystructure (left) and the predicted secondary structure (right) of theRBE portion of the A-A′ arm, the C arm and B-B′ arm of an exemplarymutated left ITR (ITR-1, left) (SEQ ID NO: 113). FIG. 3C shows theprimary structure (left) and the secondary structure (right) of theRBE-containing portion of the A-A′ loop, and the B-B′ and C-C′ arms ofwild type right AAV2 ITR (SEQ ID NO: 55). FIG. 3D shows an exemplaryright modified ITR. Shown is the primary structure (left) and thepredicted secondary structure (right) of the RBE containing portion ofthe A-A′ arm, the B-B′ and the C arm of an exemplary mutant right ITR(ITR-1, right) (SEQ ID NO: 114). Any combination of left and right ITR(e.g., AAV2 ITRs or other viral serotype or synthetic ITRs) can be usedas taught herein. Each of FIGS. 3A-3D polynucleotide sequences refer tothe sequence used in the plasmid or bacmid/baculovirus genome used toproduce the ceDNA as described herein. Also included in each of FIGS.3A-3D are corresponding ceDNA secondary structures inferred from theceDNA vector configurations in the plasmid or bacmid/baculovirus genomeand the predicted Gibbs free energy values.

FIG. 4A is a schematic illustrating an upstream process for makingbaculovirus infected insect cells (BIICs) that are useful in theproduction of a ceDNA vector for expression of the FVIII as disclosedherein in the process described in the schematic in FIG. 4B. FIG. 4B isa schematic of an exemplary method of ceDNA production and FIG. 4Cillustrates a biochemical method and process to confirm ceDNA vectorproduction. FIG. 4D and FIG. 4E are schematic illustrations describing aprocess for identifying the presence of ceDNA in DNA harvested from cellpellets obtained during the ceDNA production processes in FIG. 4B. FIG.4D shows schematic expected bands for an exemplary ceDNA either leftuncut or digested with a restriction endonuclease and then subjected toelectrophoresis on either a native gel or a denaturing gel. The leftmostschematic is a native gel, and shows multiple bands suggesting that inits duplex and uncut form ceDNA exists in at least monomeric and dimericstates, visible as a faster-migrating smaller monomer and aslower-migrating dimer that is twice the size of the monomer. Theschematic second from the left shows that when ceDNA is cut with arestriction endonuclease, the original bands are gone andfaster-migrating (e.g., smaller) bands appear, corresponding to theexpected fragment sizes remaining after the cleavage. Under denaturingconditions, the original duplex DNA is single-stranded and migrates as aspecies twice as large as observed on native gel because thecomplementary strands are covalently linked. Thus, in the secondschematic from the right, the digested ceDNA shows a similar bandingdistribution to that observed on native gel, but the bands migrate asfragments twice the size of their native gel counterparts. The rightmostschematic shows that uncut ceDNA under denaturing conditions migrates asa single-stranded open circle, and thus the observed bands are twice thesize of those observed under native conditions where the circle is notopen. In this figure “kb” is used to indicate relative size ofnucleotide molecules based, depending on context, on either nucleotidechain length (e.g., for the single stranded molecules observed indenaturing conditions) or number of base pairs (e.g., for thedouble-stranded molecules observed in native conditions). FIG. 4E showsDNA having a non-continuous structure. The ceDNA can be cut by arestriction endonuclease, having a single recognition site on the ceDNAvector, and generate two DNA fragments with different sizes (1 kb and 2kb) in both neutral and denaturing conditions. FIG. 4E also shows aceDNA having a linear and continuous structure. The ceDNA vector can becut by the restriction endonuclease and generate two DNA fragments thatmigrate as 1 kb and 2 kb in neutral conditions, but in denaturingconditions, the stands remain connected and produce single strands thatmigrate as 2 kb and 4 kb.

FIG. 5 is an exemplary picture of a denaturing gel running examples ofceDNA vectors with (+) or without (−) digestion with endonucleases(EcoRI for ceDNA construct 1 and 2; BamH1 for ceDNA construct 3 and 4;SpeI for ceDNA construct 5 and 6; and XhoI for ceDNA construct 7 and 8)Constructs 1-8 are described in Example 1 of International ApplicationPCT PCT/US18/49996, which is incorporated herein in its entirety byreference. Sizes of bands highlighted with an asterisk were determinedand provided on the bottom of the picture.

FIG. 6 depicts the results of the experiments described in Example 7 andspecifically shows the IVIS images obtained from mice treated withLNP-polyC control (mouse furthest to the left) and four mice treatedwith LNP-ceDNA-Luciferase (all but the mouse furthest to the left). Thefour ceDNA-treated mice show significant fluorescence in theliver-containing region of the mouse.

FIG. 7 depicts the results of the experiment described in Example 8. Thedark specks indicate the presence of the protein resulting from theexpressed ceDNA transgene and demonstrate association of theadministered LNP-ceDNA with hepatocytes.

FIGS. 8A and 8B depict the results of the ocular studies set forth inExample 9. FIG. 8A shows representative IVIS images fromJetPEI®-ceDNA-Luciferase-injected rat eyes (upper left) versusuninjected eye in the same rat (upper right) or plasmid-LuciferaseDNA-injected rat eye (lower left) and the uninjected eye in that samerat (lower right). FIG. 8B shows a graph of the average radianceobserved in treated eyes or the corresponding untreated eyes in each ofthe treatment groups. The ceDNA-treated rats demonstrated prolongedsignificant fluorescence (and hence luciferase transgene expression)over 99 days, in sharp contrast to rats treated with plasmid-luciferasewhere minimal relative fluorescence (and hence luciferase transgeneexpression) was observed.

FIGS. 9A and 9B depict the results of the ceDNA persistence and redosingstudy in Rag2 mice described in Example 10. FIG. 9A shows a graph oftotal flux over time observed in LNP-ceDNA-Luc-treated wild-type c57bl/6mice or Rag2 mice. FIG. 9B provides a graph showing the impact of redoseon expression levels of the luciferase transgene in Rag2 mice, withresulting increased stable expression observed after redose (arrowindicates time of redose administration).

FIG. 10 provides data from the ceDNA luciferase expression study intreated mice described in Example 11, showing total flux in each groupof mice over the duration of the study. High levels of unmethylated CpGcorrelated with lower total flux observed in the mice over time, whileuse of a liver-specific promoter correlated with durable, stableexpression of the transgene from the ceDNA vector over at least 77 days.

FIGS. 11A and 11B show hydrodynamic delivery of ceDNA vector expressingFVIII. FIG. 11A shows FVIII expression levels in serum samples at day 3and 7 from mice after hydrodynamic injection of three different ceDNAvectors expressing FVIII (LPS1-F8-v1; LPS1-F8-v2; LPS1-F8-v3), or acontrol ceDNA vector (a ceDNA expressing luciferase only) (shown asVehicle). Two of the three ceDNA vectors expressing FVIII showed FVIIIexpression. FIG. 11B is a dose-response graph of FVIII expression levelsin serum samples over a 30 day period of from mice after hydrodynamicinjection with either low or high amounts of the three different ceDNAvectors expressing FVIII (LPS1-F8-v1; LPS1-F8-v2; LPS1-F8-v3), or highamounts of the vehicle control ceDNA vector (expressing luciferaseonly).

FIG. 12 is a graph that depicts plasma FVIII concentration (IU/ml) 3days after hydrodynamic delivery of various ceDNA vectors expressingFVIII (ceDNAFVIII-vector 4, ceDNAFVIII-vector 6, ceDNAFVIII-vector 12,ceDNAFVIII-vector 14, ceDNAFVIII-vector 16, ceDNAFVIII-vector 18,ceDNAFVIII-vector 19, ceDNAFVIII-vector 21, ceDNAFVIII-vector 22).ceDNAFVIII-vector 16, ceDNAFVIII-vector 19 and ceDNAFVIII-vector 21showed the highest plasma Factor VIII concentration after 3 days.Vehicle only was used as control.

FIG. 13 is a graph that depicts plasma FVIII concentration (IU/ml) 3days after hydrodynamic delivery of various ceDNA vectors expressingFVIII (ceDNAFVIII-vector 1, ceDNAFVIII-vector 2, ceDNAFVIII-vector 3,ceDNAFVIII-vector 5, ceDNAFVIII-vector 7, ceDNAFVIII-vector 8,ceDNAFVIII-vector 12, ceDNAFVIII-vector 13, ceDNAFVIII-vector 15).ceDNAFVIII-vector 8 showed the highest plasma Factor VIII concentrationafter 3 days. Vehicle only was used as control.

FIG. 14 is a graph that depicts plasma FVIII concentration (IU/ml) 3days after hydrodynamic delivery of various ceDNA vectors expressingFVIII (ceDNAFVIII-vector 9, ceDNAFVIII-vector 10, ceDNAFVIII-vector 11,ceDNAFVIII-vector 12, ceDNAFVIII-vector 17, ceDNAFVIII-vector 20,ceDNAFVIII-vector 24, ceDNAFVIII-vector 25, ceDNAFVIII-vector 26).ceDNAFVIII-vector 17 and ceDNAFVIII-vector 20 showed the highest plasmaFactor VIII concentration after 3 days. Vehicle only was used ascontrol.

FIG. 15 is a graph that depicts plasma FVIII concentration (IU/ml) 1 dayafter hydrodynamic delivery of escalating doses of ceDNAFVIII-vector 23(0.005 μg-50 μg). As shown in FIG. 15, plasma FVIII increased in adose-dependent manner with increasing dosage of ceDNAFVIII-vector 23.Vehicle only was used as control.

FIG. 16 is a graph that depicts plasma FVIII concentration (IU/mL) at 7days after hydrodynamic delivery of the LNP:ceDNAFVIII-vector 23 testarticle.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a method for treating hemophilia A using a ceDNAvector comprising one or more nucleic acids that encode an FVIIItherapeutic protein or fragment thereof. Also provided herein are ceDNAvectors for expression of FVIII protein as described herein comprisingone or more heterologous nucleic acids that encode for the FVIIIprotein. In some embodiments, the expression of FVIII protein cancomprise secretion of the therapeutic protein out of the cell in whichit is expressed. Alternatively, in some embodiments, the expressed FVIIIprotein can act or function (e.g., exert its effect) within the cell inwhich it is expressed. In some embodiments, the ceDNA vector expressesFVIII protein in the liver, a muscle (e.g., skeletal muscle) of asubject, or other body part, which can act as a depot for FVIIItherapeutic protein production and secretion to many systemiccompartments.

I. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), FieldsVirology, 6^(th) Edition, published by Lippincott Williams & Wilkins,Philadelphia, Pa., USA (2013), Knipe, D. M. and Howley, P. M. (ed.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,A D A M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

As used herein, the terms, “administration,” “administering” andvariants thereof refers to introducing a composition or agent (e.g., atherapeutic nucleic acid or an immunosuppressant as described herein)into a subject and includes concurrent and sequential introduction ofone or more compositions or agents. “Administration” can refer, e.g., totherapeutic, pharmacokinetic, diagnostic, research, placebo, andexperimental methods. “Administration” also encompasses in vitro and exvivo treatments. The introduction of a composition or agent into asubject is by any suitable route, including orally, pulmonarily,intranasally, parenterally (intravenously, intramuscularly,intraperitoneally, or subcutaneously), rectally, intralymphatically,intratumorally, or topically. The introduction of a composition or agentinto a subject is by electroporation. Administration includesself-administration and the administration by another. Administrationcan be carried out by any suitable route. A suitable route ofadministration allows the composition or the agent to perform itsintended function. For example, if a suitable route is intravenous, thecomposition is administered by introducing the composition or agent intoa vein of the subject.

As used herein, the phrases “nucleic acid therapeutic”, “therapeuticnucleic acid” and “TNA” are used interchangeably and refer to anymodality of therapeutic using nucleic acids as an active component oftherapeutic agent to treat a disease or disorder. As used herein, thesephrases refer to RNA-based therapeutics and DNA-based therapeutics.Non-limiting examples of RNA-based therapeutics include mRNA, antisenseRNA and oligonucleotides, ribozymes, aptamers, interfering RNAs (RNAi),Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetricalinterfering RNA (aiRNA), microRNA (miRNA). Non-limiting examples ofDNA-based therapeutics include minicircle DNA, minigene, viral DNA(e.g., Lentiviral or AAV genome) or non-viral synthetic DNA vectors,closed-ended linear duplex DNA (ceDNA/CELiD), plasmids, bacmids,doggybone (dbDNA™) DNA vectors, minimalistic immunological-defined geneexpression (MIDGE)-vector, nonviral ministring DNA vector(linear-covalently closed DNA vector), or dumbbell-shaped DNA minimalvector (“dumbbell DNA”).

As used herein, an “effective amount” or “therapeutically effectiveamount” of a therapeutic agent, such as a FVIII therapeutic protein orfragment thereof, is an amount sufficient to produce the desired effect,e.g., treatment or prevention of hemophilia A. Suitable assays formeasuring expression of a target gene or target sequence include, e.g.,examination of protein or RNA levels using techniques known to those ofskill in the art such as dot blots, northern blots, in situhybridization, ELISA, immunoprecipitation, enzyme function, as well asphenotypic assays known to those of skill in the art. However, dosagelevels are based on a variety of factors, including the type of injury,the age, weight, sex, medical condition of the patient, the severity ofthe condition, the route of administration, and the particular activeagent employed. Thus, the dosage regimen may vary widely, but can bedetermined routinely by a physician using standard methods.Additionally, the terms “therapeutic amount”, “therapeutically effectiveamounts” and “pharmaceutically effective amounts” include prophylacticor preventative amounts of the compositions of the described invention.In prophylactic or preventative applications of the described invention,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of, a disease, disorder orcondition in an amount sufficient to eliminate or reduce the risk,lessen the severity, or delay the onset of the disease, disorder orcondition, including biochemical, histologic and/or behavioral symptomsof the disease, disorder or condition, its complications, andintermediate pathological phenotypes presenting during development ofthe disease, disorder or condition. It is generally preferred that amaximum dose be used, that is, the highest safe dose according to somemedical judgment. According to some embodiments, the disease, disorderor condition is hemophilia A. The terms “dose” and “dosage” are usedinterchangeably herein.

As used herein the term “therapeutic effect” refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect can include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect can also include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.

For any therapeutic agent described herein therapeutically effectiveamount may be initially determined from preliminary in vitro studiesand/or animal models. A therapeutically effective dose may also bedetermined from human data. The applied dose may be adjusted based onthe relative bioavailability and potency of the administered compound.Adjusting the dose to achieve maximal efficacy based on the methodsdescribed above and other well-known methods is within the capabilitiesof the ordinarily skilled artisan. General principles for determiningtherapeutic effectiveness, which may be found in Chapter 1 of Goodmanand Gilman's The Pharmacological Basis of Therapeutics, 10^(th) Edition,McGraw-Hill (New York) (2001), incorporated herein by reference, aresummarized below.

Pharmacokinetic principles provide a basis for modifying a dosageregimen to obtain a desired degree of therapeutic efficacy with aminimum of unacceptable adverse effects. In situations where the drug'splasma concentration can be measured and related to therapeutic window,additional guidance for dosage modification can be obtained.

As used herein, the terms “heterologous nucleotide sequence” and“transgene” are used interchangeably and refer to a nucleic acid ofinterest (other than a nucleic acid encoding a capsid polypeptide) thatis incorporated into and may be delivered and expressed by a ceDNAvector as disclosed herein.

As used herein, the terms “expression cassette” and “transcriptioncassette” are used interchangeably and refer to a linear stretch ofnucleic acids that includes a transgene that is operably linked to oneor more promoters or other regulatory sequences sufficient to directtranscription of the transgene, but which does not comprisecapsid-encoding sequences, other vector sequences or inverted terminalrepeat regions. An expression cassette may additionally comprise one ormore cis-acting sequences (e.g., promoters, enhancers, or repressors),one or more introns, and one or more post-transcriptional regulatoryelements.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includessingle, double, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNAhybrids, or a polymer including purine and pyrimidine bases or othernatural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. “Oligonucleotide” generally refers topolynucleotides of between about 5 and about 100 nucleotides of single-or double-stranded DNA. However, for the purposes of this disclosure,there is no upper limit to the length of an oligonucleotide.Oligonucleotides are also known as “oligomers” or “oligos” and may beisolated from genes, or chemically synthesized by methods known in theart. The terms “polynucleotide” and “nucleic acid” should be understoodto include, as applicable to the embodiments being described,single-stranded (such as sense or antisense) and double-strandedpolynucleotides. DNA may be in the form of, e.g., antisense molecules,plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors(P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes,chimeric sequences, chromosomal DNA, or derivatives and combinations ofthese groups. DNA may be in the form of minicircle, plasmid, bacmid,minigene, ministring DNA (linear covalently closed DNA vector),closed-ended linear duplex DNA (CELiD or ceDNA), doggybone (dbDNA™) DNA,dumbbell shaped DNA, minimalistic immunological-defined gene expression(MIDGE)-vector, viral vector or nonviral vectors. RNA may be in the formof small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpinRNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA),mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleicacids include nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, and which have similar bindingproperties as the reference nucleic acid. Examples of such analogsand/or modified residues include, without limitation, phosphorothioates,phosphorodiamidate morpholino oligomer (morpholino), phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2′-O-methylribonucleotides, locked nucleic acid (LNA™), and peptide nucleic acids(PNAs). Unless specifically limited, the term encompasses nucleic acidscontaining known analogues of natural nucleotides that have similarbinding properties as the reference nucleic acid. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions), alleles, orthologs, SNPs, and complementarysequences as well as the sequence explicitly indicated.

“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups.

“Bases” include purines and pyrimidines, which further include naturalcompounds adenine, thymine, guanine, cytosine, uracil, inosine, andnatural analogs, and synthetic derivatives of purines and pyrimidines,which include, but are not limited to, modifications which place newreactive groups such as, but not limited to, amines, alcohols, thiols,carboxylates, and alkylhalides.

As used herein, the term “interfering RNA” or “RNAi” or “interfering RNAsequence” includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides), double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA), a DNA-RNA hybrid (see, e.g., PCT Publication No. WO2004/078941), or a DNA-DNA hybrid (see, e.g., PCT Publication No. WO2004/104199) that is capable of reducing or inhibiting the expression ofa target gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule. As used herein, theterm “siRNA” includes RNA-RNA duplexes as well as DNA-RNA hybrids (see,e.g., PCT Publication No. WO 2004/078941).

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature orwhich is synthetic. The term nucleic acid construct is synonymous withthe term “expression cassette” when the nucleic acid construct containsthe control sequences required for expression of a coding sequence ofthe present disclosure. An “expression cassette” includes a DNA codingsequence operably linked to a promoter.

By “hybridizable” or “complementary” or “substantially complementary” itis meant that a nucleic acid (e.g., RNA) includes a sequence ofnucleotides that enables it to non-covalently bind, i.e. formWatson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,”to another nucleic acid in a sequence-specific, antiparallel, manner(i.e., a nucleic acid specifically binds to a complementary nucleicacid) under the appropriate in vitro and/or in vivo conditions oftemperature and solution ionic strength. As is known in the art,standard Watson-Crick base-pairing includes: adenine (A) pairing withthymidine (T), adenine (A) pairing with uracil (U), and guanine (G)pairing with cytosine (C). In addition, it is also known in the art thatfor hybridization between two RNA molecules (e.g., dsRNA), guanine (G)base pairs with uracil (U). For example, G/U base-pairing is partiallyresponsible for the degeneracy (i.e., redundancy) of the genetic code inthe context of tRNA anti-codon base-pairing with codons in mRNA. In thecontext of this disclosure, a guanine (G) of a protein-binding segment(dsRNA duplex) of a subject DNA-targeting RNA molecule is consideredcomplementary to an uracil (U), and vice versa. As such, when a G/Ubase-pair can be made at a given nucleotide position a protein-bindingsegment (dsRNA duplex) of a subject DNA-targeting RNA molecule, theposition is not considered to be non-complementary, but is insteadconsidered to be complementary.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

A DNA sequence that “encodes” a particular FVIII protein is a DNAnucleic acid sequence that is transcribed into the particular RNA and/orprotein. A DNA polynucleotide may encode an RNA (mRNA) that istranslated into protein, or a DNA polynucleotide may encode an RNA thatis not translated into protein (e.g., tRNA, rRNA, or a DNA-targetingRNA; also called “non-coding” RNA or ncRNA”).

As used herein, the term “fusion protein” as used herein refers to apolypeptide which comprises protein domains from at least two differentproteins. For example, a fusion protein may comprise (i) FVIII orfragment thereof and (ii) at least one non-GOI protein. Fusion proteinsencompassed herein include, but are not limited to, an antibody, or Fcor antigen-binding fragment of an antibody fused to a FVIII protein,e.g., an extracellular domain of a receptor, ligand, enzyme or peptide.The FVIII protein or fragment thereof that is part of a fusion proteincan be a monospecific antibody or a bispecific or multispecificantibody.

As used herein, the term “genomic safe harbor gene” or “safe harborgene” refers to a gene or loci that a nucleic acid sequence can beinserted such that the sequence can integrate and function in apredictable manner (e.g., express a protein of interest) withoutsignificant negative consequences to endogenous gene activity, or thepromotion of cancer. In some embodiments, a safe harbor gene is also aloci or gene where an inserted nucleic acid sequence can be expressedefficiently and at higher levels than a non-safe harbor site.

As used herein, the term “gene delivery” means a process by whichforeign DNA is transferred to host cells for applications of genetherapy.

As used herein, the term “terminal repeat” or “TR” includes any viralterminal repeat or synthetic sequence that comprises at least oneminimal required origin of replication and a region comprising apalindrome hairpin structure. A Rep-binding sequence (“RBS”) (alsoreferred to as RBE (Rep-binding element)) and a terminal resolution site(“TRS”) together constitute a “minimal required origin of replication”and thus the TR comprises at least one RBS and at least one TRS. TRsthat are the inverse complement of one another within a given stretch ofpolynucleotide sequence are typically each referred to as an “invertedterminal repeat” or “ITR”. In the context of a virus, ITRs mediatereplication, virus packaging, integration and provirus rescue. As wasunexpectedly found in the invention herein, TRs that are not inversecomplements across their full length can still perform the traditionalfunctions of ITRs, and thus the term ITR is used herein to refer to a TRin a ceDNA genome or ceDNA vector that is capable of mediatingreplication of ceDNA vector. It will be understood by one of ordinaryskill in the art that in complex ceDNA vector configurations more thantwo ITRs or asymmetric ITR pairs may be present. The ITR can be an AAVITR or a non-AAV ITR, or can be derived from an AAV ITR or a non-AAVITR. For example, the ITR can be derived from the family Parvoviridae,which encompasses parvoviruses and dependoviruses (e.g., canineparvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus,human parvovirus B-19), or the SV40 hairpin that serves as the origin ofSV40 replication can be used as an ITR, which can further be modified bytruncation, substitution, deletion, insertion and/or addition.Parvoviridae family viruses consist of two subfamilies Parvoviridae,which infect vertebrates, and Densovirinae, which infect invertebrates.Dependoparvoviruses include the viral family of the adeno-associatedviruses (AAV) which are capable of replication in vertebrate hostsincluding, but not limited to, human, primate, bovine, canine, equineand ovine species. For convenience herein, an ITR located 5′ to(upstream of) an expression cassette in a ceDNA vector is referred to asa “5′ ITR” or a “left ITR”, and an ITR located 3′ to (downstream of) anexpression cassette in a ceDNA vector is referred to as a “3′ ITR” or a“right ITR”.

A “wild-type ITR” or “WT-ITR” refers to the sequence of a naturallyoccurring ITR sequence in an AAV or other dependovirus that retains,e.g., Rep binding activity and Rep nicking ability. The nucleotidesequence of a WT-ITR from any AAV serotype may slightly vary from thecanonical naturally occurring sequence due to degeneracy of the geneticcode or drift, and therefore WT-ITR sequences encompassed for use hereininclude WT-ITR sequences as result of naturally occurring changes takingplace during the production process (e.g., a replication error).

As used herein, the term “substantially symmetrical WT-ITRs” or a“substantially symmetrical WT-ITR pair” refers to a pair of WT-ITRswithin a single ceDNA genome or ceDNA vector that are both wild typeITRs that have an inverse complement sequence across their entirelength. For example, an ITR can be considered to be a wild-typesequence, even if it has one or more nucleotides that deviate from thecanonical naturally occurring sequence, so long as the changes do notaffect the properties and overall three-dimensional structure of thesequence. In some aspects, the deviating nucleotides representconservative sequence changes. As one non-limiting example, a sequencethat has at least 95%, 96%, 97%, 98%, or 99% sequence identity to thecanonical sequence (as measured, e.g., using BLAST at default settings),and also has a symmetrical three-dimensional spatial organization to theother WT-ITR such that their 3D structures are the same shape ingeometrical space. The substantially symmetrical WT-ITR has the same A,C-C′ and B-B′ loops in 3D space. A substantially symmetrical WT-ITR canbe functionally confirmed as WT by determining that it has an operableRep binding site (RBE or RBE′) and terminal resolution site (TRS) thatpairs with the appropriate Rep protein. One can optionally test otherfunctions, including transgene expression under permissive conditions.

As used herein, the phrases of “modified ITR” or “mod-ITR” or “mutantITR” are used interchangeably herein and refer to an ITR that has amutation in at least one or more nucleotides as compared to the WT-ITRfrom the same serotype. The mutation can result in a change in one ormore of A, C, C′, B, B′ regions in the ITR, and can result in a changein the three-dimensional spatial organization (i.e. its 3D structure ingeometric space) as compared to the 3D spatial organization of a WT-ITRof the same serotype.

As used herein, the term “asymmetric ITRs” also referred to as“asymmetric ITR pairs” refers to a pair of ITRs within a single ceDNAgenome or ceDNA vector that are not inverse complements across theirfull length. As one non-limiting example, an asymmetric ITR pair doesnot have a symmetrical three-dimensional spatial organization to theircognate ITR such that their 3D structures are different shapes ingeometrical space. Stated differently, an asymmetrical ITR pair have thedifferent overall geometric structure, i.e., they have differentorganization of their A, C-C′ and B-B′ loops in 3D space (e.g., one ITRmay have a short C-C′ arm and/or short B-B′ arm as compared to thecognate ITR). The difference in sequence between the two ITRs may be dueto one or more nucleotide addition, deletion, truncation, or pointmutation. In one embodiment, one ITR of the asymmetric ITR pair may be awild-type AAV ITR sequence and the other ITR a modified ITR as definedherein (e.g., a non-wild-type or synthetic ITR sequence). In anotherembodiment, neither ITRs of the asymmetric ITR pair is a wild-type AAVsequence and the two ITRs are modified ITRs that have different shapesin geometrical space (i.e., a different overall geometric structure). Insome embodiments, one mod-ITRs of an asymmetric ITR pair can have ashort C-C′ arm and the other ITR can have a different modification(e.g., a single arm, or a short B-B′ arm etc.) such that they havedifferent three-dimensional spatial organization as compared to thecognate asymmetric mod-ITR.

As used herein, the term “symmetric ITRs” refers to a pair of ITRswithin a single ceDNA genome or ceDNA vector that are wild-type ormutated (e.g., modified relative to wild-type) dependoviral ITRsequences and are inverse complements across their full length. In onenon-limiting example, both ITRs are wild type ITRs sequences from AAV2.In another example, neither ITRs are wild type ITR AAV2 sequences (i.e.,they are a modified ITR, also referred to as a mutant ITR), and can havea difference in sequence from the wild type ITR due to nucleotideaddition, deletion, substitution, truncation, or point mutation. Forconvenience herein, an ITR located 5′ to (upstream of) an expressioncassette in a ceDNA vector is referred to as a “5′ ITR” or a “left ITR”,and an ITR located 3′ to (downstream of) an expression cassette in aceDNA vector is referred to as a “3′ ITR” or a “right ITR”.

As used herein, the terms “substantially symmetrical modified-ITRs” or a“substantially symmetrical mod-ITR pair” refers to a pair ofmodified-ITRs within a single ceDNA genome or ceDNA vector that are boththat have an inverse complement sequence across their entire length. Forexample, the a modified ITR can be considered substantially symmetrical,even if it has some nucleotide sequences that deviate from the inversecomplement sequence so long as the changes do not affect the propertiesand overall shape. As one non-limiting example, a sequence that has atleast 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to thecanonical sequence (as measured using BLAST at default settings), andalso has a symmetrical three-dimensional spatial organization to theircognate modified ITR such that their 3D structures are the same shape ingeometrical space. Stated differently, a substantially symmetricalmodified-ITR pair have the same A, C-C′ and B-B′ loops organized in 3Dspace. In some embodiments, the ITRs from a mod-ITR pair may havedifferent reverse complement nucleotide sequences but still have thesame symmetrical three-dimensional spatial organization—that is bothITRs have mutations that result in the same overall 3D shape. Forexample, one ITR (e.g., 5′ ITR) in a mod-ITR pair can be from oneserotype, and the other ITR (e.g., 3′ ITR) can be from a differentserotype, however, both can have the same corresponding mutation (e.g.,if the 5′ITR has a deletion in the C region, the cognate modified 3′ITRfrom a different serotype has a deletion at the corresponding positionin the C′ region), such that the modified ITR pair has the samesymmetrical three-dimensional spatial organization. In such embodiments,each ITR in a modified ITR pair can be from different serotypes (e.g.AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) such as the combination ofAAV2 and AAV6, with the modification in one ITR reflected in thecorresponding position in the cognate ITR from a different serotype. Inone embodiment, a substantially symmetrical modified ITR pair refers toa pair of modified ITRs (mod-ITRs) so long as the difference innucleotide sequences between the ITRs does not affect the properties oroverall shape and they have substantially the same shape in 3D space. Asa non-limiting example, a mod-ITR that has at least 95%, 96%, 97%, 98%or 99% sequence identity to the canonical mod-ITR as determined bystandard means well known in the art such as BLAST (Basic LocalAlignment Search Tool), or BLASTN at default settings, and also has asymmetrical three-dimensional spatial organization such that their 3Dstructure is the same shape in geometric space. A substantiallysymmetrical mod-ITR pair has the same A, C-C′ and B-B′ loops in 3Dspace, e.g., if a modified ITR in a substantially symmetrical mod-ITRpair has a deletion of a C-C′ arm, then the cognate mod-ITR has thecorresponding deletion of the C-C′ loop and also has a similar 3Dstructure of the remaining A and B-B′ loops in the same shape ingeometric space of its cognate mod-ITR.

The term “flanking” refers to a relative position of one nucleic acidsequence with respect to another nucleic acid sequence. Generally, inthe sequence ABC, B is flanked by A and C. The same is true for thearrangement A×B×C. Thus, a flanking sequence precedes or follows aflanked sequence but need not be contiguous with, or immediatelyadjacent to the flanked sequence. In one embodiment, the term flankingrefers to terminal repeats at each end of the linear duplex ceDNAvector.

As used herein, the terms “treat,” “treating,” and/or “treatment”include abrogating, substantially inhibiting, slowing or reversing theprogression of a condition, substantially ameliorating clinical symptomsof a condition, or substantially preventing the appearance of clinicalsymptoms of a condition, obtaining beneficial or desired clinicalresults. According to some embodiments, the condition is hemophilia A.Treating further refers to accomplishing one or more of the following:(a) reducing the severity of the disorder; (b) limiting development ofsymptoms characteristic of the disorder(s) being treated; (c) limitingworsening of symptoms characteristic of the disorder(s) being treated;(d) limiting recurrence of the disorder(s) in patients that havepreviously had the disorder(s); and (e) limiting recurrence of symptomsin patients that were previously asymptomatic for the disorder(s).Beneficial or desired clinical results, such as pharmacologic and/orphysiologic effects include, but are not limited to, preventing thedisease, disorder or condition from occurring in a subject that may bepredisposed to the disease, disorder or condition but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),alleviation of symptoms of the disease, disorder or condition,diminishment of extent of the disease, disorder or condition,stabilization (i.e., not worsening) of the disease, disorder orcondition, preventing spread of the disease, disorder or condition,delaying or slowing of the disease, disorder or condition progression,amelioration or palliation of the disease, disorder or condition, andcombinations thereof, as well as prolonging survival as compared toexpected survival if not receiving treatment.

As used herein, the term “increase,” “enhance,” “raise” (and like terms)generally refers to the act of increasing, either directly orindirectly, a concentration, level, function, activity, or behaviorrelative to the natural, expected, or average, or relative to a controlcondition.

As used herein, the term “minimize”, “reduce”, “decrease,” and/or“inhibit” (and like terms) generally refers to the act of reducing,either directly or indirectly, a concentration, level, function,activity, or behavior relative to the natural, expected, or average, orrelative to a control condition.

As used herein, the term “ceDNA genome” refers to an expression cassettethat further incorporates at least one inverted terminal repeat region.A ceDNA genome may further comprise one or more spacer regions. In someembodiments the ceDNA genome is incorporated as an intermolecular duplexpolynucleotide of DNA into a plasmid or viral genome.

As used herein, the term “ceDNA spacer region” refers to an interveningsequence that separates functional elements in the ceDNA vector or ceDNAgenome. In some embodiments, ceDNA spacer regions keep two functionalelements at a desired distance for optimal functionality. In someembodiments, ceDNA spacer regions provide or add to the geneticstability of the ceDNA genome within e.g., a plasmid or baculovirus. Insome embodiments, ceDNA spacer regions facilitate ready geneticmanipulation of the ceDNA genome by providing a convenient location forcloning sites and the like. For example, in certain aspects, anoligonucleotide “polylinker” containing several restriction endonucleasesites, or a non-open reading frame sequence designed to have no knownprotein (e.g., transcription factor) binding sites can be positioned inthe ceDNA genome to separate the cis—acting factors, e.g., inserting a 6mer, 12 mer, 18 mer, 24 mer, 48 mer, 86 mer, 176 mer, etc. between theterminal resolution site and the upstream transcriptional regulatoryelement Similarly, the spacer may be incorporated between thepolyadenylation signal sequence and the 3′-terminal resolution site.

As used herein, the terms “Rep binding site, “Rep binding element, “RBE”and “RBS” are used interchangeably and refer to a binding site for Repprotein (e.g., AAV Rep 78 or AAV Rep 68) which upon binding by a Repprotein permits the Rep protein to perform its site-specificendonuclease activity on the sequence incorporating the RBS. An RBSsequence and its inverse complement together form a single RBS. RBSsequences are known in the art, and include, for example,5′-GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60), an RBS sequence identified inAAV2. Any known RBS sequence may be used in the embodiments of theinvention, including other known AAV RBS sequences and other naturallyknown or synthetic RBS sequences. Without being bound by theory it isthought that he nuclease domain of a Rep protein binds to the duplexnucleotide sequence GCTC, and thus the two known AAV Rep proteins binddirectly to and stably assemble on the duplex oligonucleotide,5′-(GCGC)(GCTC)(GCTC)(GCTC)-3′ (SEQ ID NO: 60). In addition, solubleaggregated conformers (i.e., undefined number of inter-associated Repproteins) dissociate and bind to oligonucleotides that contain Repbinding sites. Each Rep protein interacts with both the nitrogenousbases and phosphodiester backbone on each strand. The interactions withthe nitrogenous bases provide sequence specificity whereas theinteractions with the phosphodiester backbone are non- or less-sequencespecific and stabilize the protein-DNA complex.

As used herein, the terms “terminal resolution site” and “TRS” are usedinterchangeably herein and refer to a region at which Rep forms atyrosine-phosphodiester bond with the 5′ thymidine generating a 3′ OHthat serves as a substrate for DNA extension via a cellular DNApolymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, theRep-thymidine complex may participate in a coordinated ligationreaction. In some embodiments, a TRS minimally encompasses anon-base-paired thymidine. In some embodiments, the nicking efficiencyof the TRS can be controlled at least in part by its distance within thesame molecule from the RBS. When the acceptor substrate is thecomplementary ITR, then the resulting product is an intramolecularduplex. TRS sequences are known in the art, and include, for example,5′-GGTTGA-3′ (SEQ ID NO: 61), the hexanucleotide sequence identified inAAV2. Any known TRS sequence may be used in the embodiments of theinvention, including other known AAV TRS sequences and other naturallyknown or synthetic TRS sequences such as AGTT (SEQ ID NO: 62), GGTTGG(SEQ ID NO: 63), AGTTGG (SEQ ID NO: 64), AGTTGA (SEQ ID NO: 65), andother motifs such as RRTTRR (SEQ ID NO: 66).

As used herein, the term “ceDNA-plasmid” refers to a plasmid thatcomprises a ceDNA genome as an intermolecular duplex.

As used herein, the term “ceDNA-bacmid” refers to an infectiousbaculovirus genome comprising a ceDNA genome as an intermolecular duplexthat is capable of propagating in E. coli as a plasmid, and so canoperate as a shuttle vector for baculovirus.

As used herein, the term “ceDNA-baculovirus” refers to a baculovirusthat comprises a ceDNA genome as an intermolecular duplex within thebaculovirus genome.

As used herein, the terms “ceDNA-baculovirus infected insect cell” and“ceDNA-BIIC” are used interchangeably, and refer to an invertebrate hostcell (including, but not limited to an insect cell (e.g., an Sf9 cell))infected with a ceDNA-baculovirus.

As used herein, the term “ceDNA” refers to capsid-free closed-endedlinear double stranded (ds) duplex DNA for non-viral gene transfer,synthetic or otherwise. Detailed description of ceDNA is described inInternational application of PCT/US2017/020828, filed Mar. 3, 2017, theentire contents of which are expressly incorporated herein by reference.Certain methods for the production of ceDNA comprising various invertedterminal repeat (ITR) sequences and configurations using cell-basedmethods are described in Example 1 of International applicationsPCT/US18/49996, filed Sep. 7, 2018, and PCT/US2018/064242, filed Dec. 6,2018 each of which is incorporated herein in its entirety by reference.Certain methods for the production of synthetic ceDNA vectors comprisingvarious ITR sequences and configurations are described, e.g., inInternational application PCT/US2019/14122, filed Jan. 18, 2019, theentire content of which is incorporated herein by reference.

As used herein, the term “closed-ended DNA vector” refers to acapsid-free DNA vector with at least one covalently closed end and whereat least part of the vector has an intramolecular duplex structure.

As used herein, the terms “ceDNA vector” and “ceDNA” are usedinterchangeably and refer to a closed-ended DNA vector comprising atleast one terminal palindrome. In some embodiments, the ceDNA comprisestwo covalently-closed ends.

As used herein, the term “neDNA” or “nicked ceDNA” refers to aclosed-ended DNA having a nick or a gap of 1-100 base pairs in a stemregion or spacer region 5′ upstream of an open reading frame (e.g., apromoter and transgene to be expressed).

As used herein, the terms “gap” and “nick” are used interchangeably andrefer to a discontinued portion of synthetic DNA vector of the presentinvention, creating a stretch of single stranded DNA portion inotherwise double stranded ceDNA. The gap can be 1 base-pair to 100base-pair long in length in one strand of a duplex DNA. Typical gaps,designed and created by the methods described herein and syntheticvectors generated by the methods can be, for example, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 bplong in length. Exemplified gaps in the present disclosure can be 1 bpto 10 bp long, 1 to 20 bp long, 1 to 30 bp long in length.

As defined herein, “reporters” refer to proteins that can be used toprovide detectable read-outs. Reporters generally produce a measurablesignal such as fluorescence, color, or luminescence. Reporter proteincoding sequences encode proteins whose presence in the cell or organismis readily observed. For example, fluorescent proteins cause a cell tofluoresce when excited with light of a particular wavelength,luciferases cause a cell to catalyze a reaction that produces light, andenzymes such as β-galactosidase convert a substrate to a coloredproduct. Exemplary reporter polypeptides useful for experimental ordiagnostic purposes include, but are not limited to β-lactamase,β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase(TK), green fluorescent protein (GFP) and other fluorescent proteins,chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art.

As used herein, the terms “sense” and “antisense” refer to theorientation of the structural element on the polynucleotide. The senseand antisense versions of an element are the reverse complement of eachother.

As used herein, the term “synthetic AAV vector” and “syntheticproduction of AAV vector” refers to an AAV vector and syntheticproduction methods thereof in an entirely cell-free environment.

As used herein, “reporters” refer to proteins that can be used toprovide detectable read-outs. Reporters generally produce a measurablesignal such as fluorescence, color, or luminescence. Reporter proteincoding sequences encode proteins whose presence in the cell or organismis readily observed. For example, fluorescent proteins cause a cell tofluoresce when excited with light of a particular wavelength,luciferases cause a cell to catalyze a reaction that produces light, andenzymes such as β-galactosidase convert a substrate to a coloredproduct. Exemplary reporter polypeptides useful for experimental ordiagnostic purposes include, but are not limited to β-lactamase,β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase(TK), green fluorescent protein (GFP) and other fluorescent proteins,chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art.

As used herein, the term “effector protein” refers to a polypeptide thatprovides a detectable read-out, either as, for example, a reporterpolypeptide, or more appropriately, as a polypeptide that kills a cell,e.g., a toxin, or an agent that renders a cell susceptible to killingwith a chosen agent or lack thereof. Effector proteins include anyprotein or peptide that directly targets or damages the host cell's DNAand/or RNA. For example, effector proteins can include, but are notlimited to, a restriction endonuclease that targets a host cell DNAsequence (whether genomic or on an extrachromosomal element), a proteasethat degrades a polypeptide target necessary for cell survival, a DNAgyrase inhibitor, and a ribonuclease-type toxin. In some embodiments,the expression of an effector protein controlled by a syntheticbiological circuit as described herein can participate as a factor inanother synthetic biological circuit to thereby expand the range andcomplexity of a biological circuit system's responsiveness.

Transcriptional regulators refer to transcriptional activators andrepressors that either activate or repress transcription of a gene ofinterest, such as FVIII. Promoters are regions of nucleic acid thatinitiate transcription of a particular gene Transcriptional activatorstypically bind nearby to transcriptional promoters and recruit RNApolymerase to directly initiate transcription. Repressors bind totranscriptional promoters and sterically hinder transcriptionalinitiation by RNA polymerase. Other transcriptional regulators may serveas either an activator or a repressor depending on where they bind andcellular and environmental conditions. Non-limiting examples oftranscriptional regulator classes include, but are not limited tohomeodomain proteins, zinc-finger proteins, winged-helix (forkhead)proteins, and leucine-zipper proteins.

As used herein, a “repressor protein” or “inducer protein” is a proteinthat binds to a regulatory sequence element and represses or activates,respectively, the transcription of sequences operatively linked to theregulatory sequence element. Preferred repressor and inducer proteins asdescribed herein are sensitive to the presence or absence of at leastone input agent or environmental input. Preferred proteins as describedherein are modular in form, comprising, for example, separableDNA-binding and input agent-binding or responsive elements or domains.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutically active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce a toxic, anallergic, or similar untoward reaction when administered to a host.

As used herein, an “input agent responsive domain” is a domain of atranscription factor that binds to or otherwise responds to a conditionor input agent in a manner that renders a linked DNA binding fusiondomain responsive to the presence of that condition or input. In oneembodiment, the presence of the condition or input results in aconformational change in the input agent responsive domain, or in aprotein to which it is fused, that modifies the transcription-modulatingactivity of the transcription factor.

The term “in vivo” refers to assays or processes that occur in or withinan organism, such as a multicellular animal In some of the aspectsdescribed herein, a method or use can be said to occur “in vivo” when aunicellular organism, such as a bacterium, is used. The term “ex vivo”refers to methods and uses that are performed using a living cell withan intact membrane that is outside of the body of a multicellular animalor plant, e.g., explants, cultured cells, including primary cells andcell lines, transformed cell lines, and extracted tissue or cells,including blood cells, among others. The term “in vitro” refers toassays and methods that do not require the presence of a cell with anintact membrane, such as cellular extracts, and can refer to theintroducing of a programmable synthetic biological circuit in anon-cellular system, such as a medium not comprising cells or cellularsystems, such as cellular extracts.

The term “promoter,” as used herein, refers to any nucleic acid sequencethat regulates the expression of another nucleic acid sequence bydriving transcription of the nucleic acid sequence, which can be aheterologous target gene encoding a protein or an RNA. Promoters can beconstitutive, inducible, repressible, tissue-specific, or anycombination thereof. A promoter is a control region of a nucleic acidsequence at which initiation and rate of transcription of the remainderof a nucleic acid sequence are controlled. A promoter can also containgenetic elements at which regulatory proteins and molecules can bind,such as RNA polymerase and other transcription factors. In someembodiments of the aspects described herein, a promoter can drive theexpression of a transcription factor that regulates the expression ofthe promoter itself. Within the promoter sequence will be found atranscription initiation site, as well as protein binding domainsresponsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain “TATA” boxes and “CAT” boxes. Variouspromoters, including inducible promoters, may be used to drive theexpression of transgenes in the ceDNA vectors disclosed herein. Apromoter sequence may be bounded at its 3′ terminus by the transcriptioninitiation site and extends upstream (5′ direction) to include theminimum number of bases or elements necessary to initiate transcriptionat levels detectable above background.

The term “enhancer” as used herein refers to a cis-acting regulatorysequence (e.g., 50-1,500 base pairs) that binds one or more proteins(e.g., activator proteins, or transcription factor) to increasetranscriptional activation of a nucleic acid sequence. Enhancers can bepositioned up to 1,000,000 base pars upstream of the gene start site ordownstream of the gene start site that they regulate. An enhancer can bepositioned within an intronic region, or in the exonic region of anunrelated gene.

A promoter can be said to drive expression or drive transcription of thenucleic acid sequence that it regulates. The phrases “operably linked,”“operatively positioned,” “operatively linked,” “under control,” and“under transcriptional control” indicate that a promoter is in a correctfunctional location and/or orientation in relation to a nucleic acidsequence it regulates to control transcriptional initiation and/orexpression of that sequence. An “inverted promoter,” as used herein,refers to a promoter in which the nucleic acid sequence is in thereverse orientation, such that what was the coding strand is now thenon-coding strand, and vice versa. Inverted promoter sequences can beused in various embodiments to regulate the state of a switch. Inaddition, in various embodiments, a promoter can be used in conjunctionwith an enhancer.

A promoter can be one naturally associated with a gene or sequence, ascan be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon of a given gene or sequence.Such a promoter can be referred to as “endogenous.” Similarly, in someembodiments, an enhancer can be one naturally associated with a nucleicacid sequence, located either downstream or upstream of that sequence.

In some embodiments, a coding nucleic acid segment is positioned underthe control of a “recombinant promoter” or “heterologous promoter,” bothof which refer to a promoter that is not normally associated with theencoded nucleic acid sequence it is operably linked to in its naturalenvironment. A recombinant or heterologous enhancer refers to anenhancer not normally associated with a given nucleic acid sequence inits natural environment. Such promoters or enhancers can includepromoters or enhancers of other genes; promoters or enhancers isolatedfrom any other prokaryotic, viral, or eukaryotic cell; and syntheticpromoters or enhancers that are not “naturally occurring,” i.e.,comprise different elements of different transcriptional regulatoryregions, and/or mutations that alter expression through methods ofgenetic engineering that are known in the art. In addition to producingnucleic acid sequences of promoters and enhancers synthetically,promoter sequences can be produced using recombinant cloning and/ornucleic acid amplification technology, including PCR, in connection withthe synthetic biological circuits and modules disclosed herein (see,e.g., U.S. Pat. Nos. 4,683,202, 5,928,906, each incorporated herein byreference). Furthermore, it is contemplated that control sequences thatdirect transcription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

As described herein, an “inducible promoter” is one that ischaracterized by initiating or enhancing transcriptional activity whenin the presence of, influenced by, or contacted by an inducer orinducing agent. An “inducer” or “inducing agent,” as defined herein, canbe endogenous, or a normally exogenous compound or protein that isadministered in such a way as to be active in inducing transcriptionalactivity from the inducible promoter. In some embodiments, the induceror inducing agent, i.e., a chemical, a compound or a protein, can itselfbe the result of transcription or expression of a nucleic acid sequence(i.e., an inducer can be an inducer protein expressed by anothercomponent or module), which itself can be under the control or aninducible promoter. In some embodiments, an inducible promoter isinduced in the absence of certain agents, such as a repressor. Examplesof inducible promoters include but are not limited to, tetracycline,metallothionine, ecdysone, mammalian viruses (e.g., the adenovirus latepromoter; and the mouse mammary tumor virus long terminal repeat(MMTV-LTR)) and other steroid-responsive promoters, rapamycin responsivepromoters and the like.

The terms “DNA regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate transcription ofa non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence(e.g., site-directed modifying polypeptide, or Cas9/Csn1 polypeptide)and/or regulate translation of an encoded polypeptide.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression. An“expression cassette” includes a heterologous DNA sequence that isoperably linked to a promoter or other regulatory sequence sufficient todirect transcription of the transgene in the ceDNA vector. Suitablepromoters include, for example, tissue specific promoters. Promoters canalso be of AAV origin.

The term “subject” as used herein refers to a human or animal, to whomtreatment, including prophylactic treatment, with the ceDNA vectoraccording to the present invention, is provided. Usually the animal is avertebrate such as, but not limited to a primate, rodent, domesticanimal or game animal Primates include but are not limited to,chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g.,Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits andhamsters. Domestic and game animals include, but are not limited to,cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domesticcat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken,emu, ostrich, and fish, e.g., trout, catfish and salmon. In certainembodiments of the aspects described herein, the subject is a mammal,e.g., a primate or a human A subject can be male or female.Additionally, a subject can be an infant or a child. In someembodiments, the subject can be a neonate or an unborn subject, e.g.,the subject is in utero. Preferably, the subject is a mammal. The mammalcan be a human, non-human primate, mouse, rat, dog, cat, horse, or cow,but is not limited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of diseasesand disorders. In addition, the methods and compositions describedherein can be used for domesticated animals and/or pets. A human subjectcan be of any age, gender, race or ethnic group, e.g., Caucasian(white), Asian, African, black, African American, African European,Hispanic, Mideastern, etc. In some embodiments, the subject can be apatient or other subject in a clinical setting. In some embodiments, thesubject is already undergoing treatment. In some embodiments, thesubject is an embryo, a fetus, neonate, infant, child, adolescent, oradult. In some embodiments, the subject is a human fetus, human neonate,human infant, human child, human adolescent, or human adult. In someembodiments, the subject is an animal embryo, or non-human embryo ornon-human primate embryo. In some embodiments, the subject is a humanembryo.

As used herein, the term “host cell”, includes any cell type that issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid construct or ceDNA expression vector of the presentdisclosure. As non-limiting examples, a host cell can be an isolatedprimary cell, pluripotent stem cells, CD34⁺ cells), induced pluripotentstem cells, or any of a number of immortalized cell lines (e.g., HepG2cells). Alternatively, a host cell can be an in situ or in vivo cell ina tissue, organ or organism.

The term “exogenous” refers to a substance present in a cell other thanits native source. The term “exogenous” when used herein can refer to anucleic acid (e.g., a nucleic acid encoding a polypeptide) or apolypeptide that has been introduced by a process involving the hand ofman into a biological system such as a cell or organism in which it isnot normally found and one wishes to introduce the nucleic acid orpolypeptide into such a cell or organism. Alternatively, “exogenous” canrefer to a nucleic acid or a polypeptide that has been introduced by aprocess involving the hand of man into a biological system such as acell or organism in which it is found in relatively low amounts and onewishes to increase the amount of the nucleic acid or polypeptide in thecell or organism, e.g., to create ectopic expression or levels. Incontrast, the term “endogenous” refers to a substance that is native tothe biological system or cell.

The term “sequence identity” refers to the relatedness between twonucleotide sequences. For purposes of the present disclosure, the degreeof sequence identity between two deoxyribonucleotide sequences isdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, supra), preferably version 3.0.0 or later. The optionalparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitutionmatrix. The output of Needle labeled “longest identity” (obtained usingthe -nobrief option) is used as the percent identity and is calculatedas follows: (Identical Deoxyribonucleotides.times.100)/(Length ofAlignment-Total Number of Gaps in Alignment). The length of thealignment is preferably at least 10 nucleotides, preferably at least 25nucleotides more preferred at least 50 nucleotides and most preferred atleast 100 nucleotides.

The term “homology” or “homologous” as used herein is defined as thepercentage of nucleotide residues that are identical to the nucleotideresidues in the corresponding sequence on the target chromosome, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleotide sequence homology can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ClustalW2 or Megalign (DNASTAR) software. Those skilled in the art candetermine appropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. In some embodiments, a nucleic acidsequence (e.g., DNA sequence), for example of a homology arm, isconsidered “homologous” when the sequence is at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more, identical to the corresponding nativeor unedited nucleic acid sequence (e.g., genomic sequence) of the hostcell.

The term “heterologous,” as used herein, means a nucleotide orpolypeptide sequence that is not found in the native nucleic acid orprotein, respectively. A heterologous nucleic acid sequence may belinked to a naturally-occurring nucleic acid sequence (or a variantthereof) (e.g., by genetic engineering) to generate a chimericnucleotide sequence encoding a chimeric polypeptide. A heterologousnucleic acid sequence may be linked to a variant polypeptide (e.g., bygenetic engineering) to generate a nucleotide sequence encoding a fusionvariant polypeptide.

A “vector” or “expression vector” is a replicon, such as plasmid,bacmid, phage, virus, virion, or cosmid, to which another DNA segment,i.e. an “insert”, may be attached so as to bring about the replicationof the attached segment in a cell. A vector can be a nucleic acidconstruct designed for delivery to a host cell or for transfer betweendifferent host cells. As used herein, a vector can be viral or non-viralin origin and/or in final form, however for the purpose of the presentdisclosure, a “vector” generally refers to a ceDNA vector, as that termis used herein. The term “vector” encompasses any genetic element thatis capable of replication when associated with the proper controlelements and that can transfer gene sequences to cells. In someembodiments, a vector can be an expression vector or recombinant vector.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g., 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra chromosomal DNAthereby eliminating potential effects of chromosomal integration.

The phrase “genetic disease” as used herein refers to a disease,partially or completely, directly or indirectly, caused by one or moreabnormalities in the genome, especially a condition that is present frombirth. The abnormality may be a mutation, an insertion or a deletion.The abnormality may affect the coding sequence of the gene or itsregulatory sequence. The genetic disease may be, but not limited to DMD,hemophilia, cystic fibrosis, Huntington's chorea, familialhypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson'sdisease, congenital hepatic porphyria, inherited disorders of hepaticmetabolism, Lesch Nyhan syndrome, sickle cell anemia, thalassemia,xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxiatelangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease.

An “inhibitory polynucleotide” as used herein refers to a DNA or RNAmolecule that reduces or prevents expression (transcription ortranslation) of a second (target) polynucleotide. Inhibitorypolynucleotides include antisense polynucleotides, ribozymes, andexternal guide sequences. The term “inhibitory polynucleotide” furtherincludes DNA and RNA molecules, e.g., RNAi that encode the actualinhibitory species, such as DNA molecules that encode ribozymes.

As used herein, “gene silencing” or “gene silenced” in reference to anactivity of an RNAi molecule, for example a siRNA or miRNA refers to adecrease in the mRNA level in a cell for a target gene.

As used herein, the term “RNAi” refers to any type of interfering RNA,including but not limited to, siRNA, shRNAi, endogenous microRNA andartificial microRNA. For instance, it includes sequences previouslyidentified as siRNA, regardless of the mechanism of down-streamprocessing of the RNA (i.e. although siRNAs are believed to have aspecific method of in vivo processing resulting in the cleavage of mRNA,such sequences can be incorporated into the vectors in the context ofthe flanking sequences described herein). The term “RNAi” can includeboth gene silencing RNAi molecules, and also RNAi effector moleculeswhich activate the expression of a gene.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment. The use of “comprising”indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth Similarly, the word “or”is intended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to limit thescope of the present invention, which is defined solely by the claims.

II. Expression of a FVIII Protein from a ceDNA Vector

The technology described herein is directed in general to the expressionand/or production of FVIII protein in a cell from a non-viral DNAvector, e.g., a ceDNA vector as described herein. ceDNA vectors forexpression of FVIII protein are described herein in the section entitled“ceDNA vectors in general”. In particular, ceDNA vectors for expressionof FVIII protein comprise a pair of ITRs (e.g., symmetric or asymmetricas described herein) and between the ITR pair, a nucleic acid encodingan FVIII protein, as described herein, operatively linked to a promoteror regulatory sequence. A distinct advantage of ceDNA vectors forexpression of FVIII protein over traditional AAV vectors, and evenlentiviral vectors, is that there is no size constraint for theheterologous nucleic acid sequences encoding a desired protein. Thus,even a full length 6.8 kb FVIII protein can be expressed from a singleceDNA vector. Thus, the ceDNA vectors described herein can be used toexpress a therapeutic FVIII protein in a subject in need thereof, e.g.,a subject with hemophilia A.

As one will appreciate, the ceDNA vector technologies described hereincan be adapted to any level of complexity or can be used in a modularfashion, where expression of different components of a FVIII protein canbe controlled in an independent manner. For example, it is specificallycontemplated that the ceDNA vector technologies designed herein can beas simple as using a single ceDNA vector to express a singleheterologous gene sequence (e.g., a FVIII protein) or can be as complexas using multiple ceDNA vectors, where each vector expresses multipleFVIII proteins or associated co-factors or accessory proteins that areeach independently controlled by different promoters. The followingembodiments are specifically contemplated herein and can adapted by oneof skill in the art as desired.

In one embodiment, a single ceDNA vector can be used to express a singlecomponent of an FVIII protein. Alternatively, a single ceDNA vector canbe used to express multiple components (e.g., at least 2) of a FVIIIprotein under the control of a single promoter (e.g., a strongpromoter), optionally using an IRES sequence(s) to ensure appropriateexpression of each of the components, e.g., co-factors or accessoryproteins.

Also contemplated herein, in another embodiment, is a single ceDNAvector comprising at least two inserts (e.g., expressing a heavy chainor light chain), where the expression of each insert is under thecontrol of its own promoter. The promoters can include multiple copiesof the same promoter, multiple different promoters, or any combinationthereof. As one of skill in the art will appreciate, it is oftendesirable to express components of a FVIII protein at differentexpression levels, thus controlling the stoichiometry of the individualcomponents expressed to ensure efficient a FVIII protein folding andcombination in the cell.

Additional variations of ceDNA vector technologies can be envisioned byone of skill in the art or can be adapted from protein productionmethods using conventional vectors.

A. Nucleic Acids

The characterization and development of nucleic acid molecules forpotential therapeutic use are provided herein. According to someembodiments, the nucleic acids for therapeutic use encode a FVIIIprotein. In some embodiments, chemical modification of oligonucleotidesfor the purpose of altered and improved in vivo properties (delivery,stability, lifetime, folding, target specificity), as well as theirbiological function and mechanism that directly correlate withtherapeutic application, are described where appropriate.

Illustrative therapeutic nucleic acids of the present disclosure thatcan be immunostimulatory and require use of immunosuppressants disclosedherein can include, but are not limited to, minigenes, plasmids,minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisenseoligonucleotides (ASO), ribozymes, closed ended double stranded DNA(e.g., ceDNA, CELiD, linear covalently closed DNA (“ministring”),doggybone (dbDNA™), protelomere closed ended DNA, or dumbbell linearDNA), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetricalinterfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, and DNAviral vectors, viral RNA vector, and any combination thereof.

siRNA or miRNA that can downregulate the intracellular levels ofspecific proteins through a process called RNA interference (RNAi) arealso contemplated by the present invention to be nucleic acidtherapeutics. After siRNA or miRNA is introduced into the cytoplasm of ahost cell, these double-stranded RNA constructs can bind to a proteincalled RISC. The sense strand of the siRNA or miRNA is removed by theRISC complex. The RISC complex, when combined with the complementarymRNA, cleaves the mRNA and release the cut strands. RNAi is by inducingspecific destruction of mRNA that results in downregulation of acorresponding protein.

Antisense oligonucleotides (ASO) and ribozymes that inhibit mRNAtranslation into protein can be nucleic acid therapeutics. For antisenseconstructs, these single stranded deoxy nucleic acids have acomplementary sequence to the sequence of the target protein mRNA, andWatson—capable of binding to the mRNA by Crick base pairing. Thisbinding prevents translation of a target mRNA, and/or triggers RNaseHdegradation of the mRNA transcript. As a result, the antisenseoligonucleotide has increased specificity of action (i.e.,down-regulation of a specific disease-related protein).

In any of the methods provided herein, the therapeutic nucleic acid canbe a therapeutic RNA. The therapeutic RNA can be an inhibitor of mRNAtranslation, agent of RNA interference (RNAi), catalytically active RNAmolecule (ribozyme), transfer RNA (tRNA) or an RNA that binds an mRNAtranscript (ASO), protein or other molecular ligand (aptamer). In any ofthe methods provided herein, the agent of RNAi can be a double-strandedRNA, single-stranded RNA, micro RNA, short interfering RNA, shorthairpin RNA, or a triplex-forming oligonucleotide.

According to some embodiments, the therapeutic nucleic acid is a closedended double stranded DNA, e.g., a ceDNA. According to some embodiments,the expression and/or production of a therapeutic protein in a cell isfrom a non-viral DNA vector, e.g., a ceDNA vector. A distinct advantageof ceDNA vectors for expression of a therapeutic protein overtraditional AAV vectors, and even lentiviral vectors, is that there isno size constraint for the heterologous nucleic acid sequences encodinga desired protein. Thus, even a large therapeutic protein can beexpressed from a single ceDNA vector. Thus, ceDNA vectors can be used toexpress a therapeutic protein in a subject in need thereof.

In general, a ceDNA vector for expression of a therapeutic protein asdisclosed herein, comprises in the 5′ to 3′ direction: a firstadeno-associated virus (AAV) inverted terminal repeat (ITR), anucleotide sequence of interest (for example an expression cassette asdescribed herein) and a second AAV ITR. The ITR sequences selected fromany of: (i) at least one WT ITR and at least one modified AAV invertedterminal repeat (mod-ITR) (e.g., asymmetric modified ITRs); (ii) twomodified ITRs where the mod-ITR pair have a different three-dimensionalspatial organization with respect to each other (e.g., asymmetricmodified ITRs), or (iii) symmetrical or substantially symmetrical WT-WTITR pair, where each WT-ITR has the same three-dimensional spatialorganization, or (iv) symmetrical or substantially symmetrical modifiedITR pair, where each mod-ITR has the same three-dimensional spatialorganization.

In some embodiments, a transgene encoding the FVIII protein can alsoencode a secretory sequence so that the a FVIII protein is directed tothe Golgi Apparatus and Endoplasmic Reticulum whence a FVIII proteinwill be folded into the correct conformation by chaperone molecules asit passes through the ER and out of the cell. Exemplary secretorysequences include, but are not limited to VH-02 (SEQ ID NO: 88) andVK-A26 (SEQ ID NO: 89) and Igκ signal sequence (SEQ ID NO: 126), as wellas a Gluc secretory signal that allows the tagged protein to be secretedout of the cytosol (SEQ ID NO: 188), TMD-ST secretory sequence, thatdirects the tagged protein to the Golgi (SEQ ID NO: 189).

Regulatory switches can also be used to fine tune the expression of theFVIII protein so that the FVIII protein is expressed as desired,including but not limited to expression of the FVIII protein at adesired expression level or amount, or alternatively, when there is thepresence or absence of particular signal, including a cellular signalingevent. For instance, as described herein, expression of the FVIIIprotein from the ceDNA vector can be turned on or turned off when aparticular condition occurs, as described herein in the section entitledRegulatory Switches.

For example, and for illustration purposes only, FVIII proteins can beused to turn off undesired reaction, such as too high a level ofproduction of the FVIII protein. The FVIII gene can contain a signalpeptide marker to bring the FVIII protein to the desired cell. However,in either situation it can be desirable to regulate the expression ofthe FVIII protein. ceDNA vectors readily accommodate the use ofregulatory switches.

A distinct advantage of ceDNA vectors over traditional AAV vectors, andeven lentiviral vectors, is that there is no size constraint for theheterologous nucleic acid sequences encoding the FVIII protein. Thus,even a full length FVIII, as well as optionally any co-factors orassessor proteins can be expressed from a single ceDNA vector. Inaddition, depending on the necessary stoichiometry one can expressmultiple segments of the same FVIII protein, and can use same ordifferent promoters, and can also use regulatory switches to fine tuneexpression of each region. For example, as shown in the Examples, aceDNA vector that comprises a dual promoter system can be used, so thata different promoter is used for each domain of the FVIII protein. Useof a ceDNA plasmid to produce the FVIII protein can include a uniquecombination of promoters for expression of the domains of the FVIIIprotein that results in the proper ratios of each domain for theformation of functional FVIII protein. Accordingly, in some embodiments,a ceDNA vector can be used to express different regions of FVIII proteinseparately (e.g., under control of a different promoter).

In another embodiment, the FVIII protein expressed from the ceDNAvectors further comprises an additional functionality, such asfluorescence, enzyme activity, secretion signal or immune cellactivator.

In some embodiments, the ceDNA encoding the FVIII protein can furthercomprise a linker domain, for example. As used herein “linker domain”refers to an oligo- or polypeptide region from about 2 to 100 aminoacids in length, which links together any of the domains/regions of thea FVIII protein as described herein. In some embodiment, linkers caninclude or be composed of flexible residues such as glycine and serineso that the adjacent protein domains are free to move relative to oneanother. Longer linkers may be used when it is desirable to ensure thattwo adjacent domains do not sterically interfere with one another.Linkers may be cleavable or non-cleavable. Examples of cleavable linkersinclude 2A linkers (for example T2A), 2A-like linkers or functionalequivalents thereof and combinations thereof. The linker can be a linkerregion is T2A derived from Thosea asigna virus.

It is well within the abilities of one of skill in the art to take aknown and/or publicly available protein sequence of e.g., the FVIIIetc., and reverse engineer a cDNA sequence to encode such a protein. ThecDNA can then be codon optimized to match the intended host cell andinserted into a ceDNA vector as described herein.

B. ceDNA Vectors Expressing FVIII Protein

A ceDNA vector for expression of FVIII protein having one or moresequences encoding a desired FVIII can comprise regulatory sequencessuch as promoters, secretion signals, polyA regions, and enhancers. At aminimum, a ceDNA vector comprises one or more heterologous sequencesencoding a FVIII protein.

In order to achieve highly efficient and accurate FVIII proteinassembly, it is specifically contemplated in some embodiments that theFVIII protein comprise an endoplasmic reticulum ER leader sequence todirect it to the ER, where protein folding occurs. For example, asequence that directs the expressed protein(s) to the ER for folding.

In some embodiments, a cellular or extracellular localization signal(e.g., secretory signal, nuclear localization signal, mitochondriallocalization signal etc.) is comprised in the ceDNA vector to direct thesecretion or desired subcellular localization of FVIII such that theFVIII protein can bind to intracellular target(s) (e.g., an intrabody)or extracellular target(s).

In some embodiments, a ceDNA vector for expression of FVIII protein asdescribed herein permits the assembly and expression of any desiredFVIII protein in a modular fashion. As used herein, the term “modular”refers to elements in a ceDNA expressing plasmid that can be readilyremoved from the construct. For example, modular elements in aceDNA-generating plasmid comprise unique pairs of restriction sitesflanking each element within the construct, enabling the exclusivemanipulation of individual elements (see e.g., FIGS. 1A-1G). Thus, theceDNA vector platform can permit the expression and assembly of anydesired FVIII protein configuration. Provided herein in variousembodiments are ceDNA plasmid vectors that can reduce and/or minimizethe amount of manipulation required to assemble a desired ceDNA vectorencoding FVIII protein.

C. Exemplary FVIII Proteins Expressed by ceDNA Vectors

In particular, a ceDNA vector for expression of FVIII protein asdisclosed herein can encode, for example, but is not limited to, FVIIIproteins, as well as variants, and/or active fragments thereof, for usein the treatment, prophylaxis, and/or amelioration of one or moresymptoms of hemophilia A. In one aspect, the hemophilia A is a humanhemophilia A.

(i) FVIII therapeutic proteins and fragments thereof

Essentially any version of the FVIII therapeutic protein or fragmentthereof (e.g., functional fragment) can be encoded by and expressed inand from a ceDNA vector as described herein. One of skill in the artwill understand that FVIII therapeutic protein includes all splicevariants and orthologs of the FVIII protein. FVIII therapeutic proteinincludes intact molecules as well as fragments (e.g., functional)thereof.

Factor VIII

Factor VIII is the nonenzymatic cofactor to the activated clottingfactor IX (FIXa), which, when proteolytically activated, interacts withFIXa to form a tight noncovalent complex that binds to and activatesfactor X (FX).

The Factor VIII gene or protein can also be referred to as F8,Coagulation Factor VIII, Procoagulant Component, Antihemophilic Factor,F8C, AHF, DXS1253E, FVIII, HEMA, or F8B. Expression of the Factor VIIIgene is tissue-specific and is mostly observed in liver cells. Thehighest level of the mRNA and Factor VIII proteins has been detected inliver sinusoidal cells; significant amounts of Factor VIII are alsopresent in hepatocytes and in Kupffer cells (resident macrophages ofliver sinusoids). Moderate levels of Factor VIII protein are detectablein the serum and plasma. Low to moderate levels of Factor VIII proteinare expressed in fetal brain, retina, kidney and testis.

Factor VIII mRNA is expressed throughout many tissues of the body,including bone marrow, whole blood, white blood cells, lymph nodes,thymus, brain, cerebral cortex, cerebellum, retina, spinal cord, tibialnerve, heart, artery, smooth muscle, skeletal muscle, small intestine,colon, adipocytes, kidney, liver, lung, spleen, stomach, esophagus,bladder, pancreas, thyroid, salivary gland, adrenal gland, pituitarygland, breast, skin, ovary, uterus, placenta, prostate, and testis. TheFVIII gene localized on the long arm of the X chromosome occupies aregion approximately 186 kbp long and consists of 26 exons (69-3,106 bp)and introns (from 207 bp to 32.4 kbp). The total length of the codingsequence of this gene is 9 kbp.

The mature factor VIII polypeptide comprises the A1-A2-B-A3-C1-C2structural domains. Three acidic subdomains, which are denoted asa1-a3-A1(a1)-A2(a2)-B-(a3)A3-C1-C2, localize at the boundaries of Adomains and play a significant role in the interaction between FVIII andother proteins (in particular, with thrombin). Mutations in thesesubdomains reduce the level of factor VIII activation by thrombin.

The factor VIII protein (Coagulation factor VIII isoform) is apreproprotein [Homo sapiens]; Accession number: NP_000123.1 (2351 aa)and has the following sequence:

(SEQ ID NO: 218) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

A distinct advantage of ceDNA vectors over traditional AAV vectors, andeven lentiviral vectors, is that there is no size constraint for theheterologous nucleic acid sequences encoding a desired protein. Thus,multiple full length FVIII therapeutic proteins can be expressed from asingle ceDNA vector.

Expression of FVIII therapeutic protein or fragment thereof from a ceDNAvector can be achieved both spatially and temporally using one or moreinducible or repressible promoters, as known in the art or describedherein, including regulatory switches as described herein.

In one embodiment, FVIII therapeutic protein is an “therapeutic proteinvariant,” which refers to the FVIII therapeutic protein having analtered amino acid sequence, composition or structure as compared to itscorresponding native FVIII therapeutic protein. In one embodiment, FVIIIis a functional version (e.g., wild type). It may also be useful toexpress a mutant version of FVIII protein such as a point mutation ordeletion mutation that leads to hemophilia A, e.g., for an animal modelof the disease and/or for assessing drugs for hemophilia A. Delivery ofmutant or modified FVIII proteins to a cell or animal model system canbe done in order to generate a disease model. Such a cellular or animalmodel can be used for research and/or drug screening. FVIII therapeuticprotein expressed from the ceDNA vectors may further comprise asequence/moiety that confers an additional functionality, such asfluorescence, enzyme activity, or secretion signal. In one embodiment,an FVIII therapeutic protein variant comprises a non-native tag sequencefor identification (e.g., an immunotag) to allow it to be distinguishedfrom endogenous FVIII therapeutic protein in a recipient host cell.

It is well within the abilities of one of skill in the art to take aknown and/or publicly available protein sequence of e.g., FVIIItherapeutic protein and reverse engineer a cDNA sequence to encode sucha protein. The cDNA can then be codon optimized to match the intendedhost cell and inserted into a ceDNA vector as described herein.

In one embodiment, the FVIII therapeutic protein encoding sequence canbe derived from an existing host cell or cell line, for example, byreverse transcribing mRNA obtained from the host and amplifying thesequence using PCR.

(ii) FVIII Therapeutic Protein Expressing ceDNA Vectors

A ceDNA vector having one or more sequences encoding a desired FVIIItherapeutic protein can comprise regulatory sequences such as promoters,secretion signals, polyA regions, and enhancers. At a minimum, a ceDNAvector comprises one or more heterologous sequences encoding the FVIIItherapeutic protein or functional fragment thereof. Exemplary cassetteinserts for generating ceDNA vectors encoding the FVIII therapeuticproteins are depicted in FIGS. 1A-1G. In one embodiment, the ceDNAvector comprises an FVIII sequence listed in Table 1 herein.

TABLE 1 Exemplary FVIII sequences for treatment of hemophilia AExemplary nucleic acid sequences encoding Factor VIII SEQ Ref- CG IDDescription Length erence Content NO: Sequence B domain-deleted 4374https://   3 380ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGG(BDD) hEVIII with www.ncbi.GGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTSQ sequence I nlm.nih.GCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACAgov/TTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACpubmed/CCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGA29292164GTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAACCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGAB domain-deleted 4374 US2017   0 381ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGTCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGGTATTACCTGGG(BDD) hEVIII with 0216408GGCTGTGGAGCTGAGCTGGGACTATATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCTAGGTTCCCCCCCAGGGTSQ sequence II A1GCCCAAGAGCTTCCCCTTTAACACTTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGGCTGCTGGGGCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCTTCTGAGGGGGCTGAGTATGATGACCAGACTAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTTCCTGGGGGCAGCCATACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGTCTCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCTCTGCTGGTGTGTAGGGAGGGCAGCCTGGCTAAGGAAAAGACCCAGACCCTGCATAAGTTTATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAGACCAAGAACAGCCTGATGCAGGATAGGGATGCTGCCTCTGCCAGGGCTGGCCTAAGATGCACACTGTGAATGGGTATGTGAATAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGGATGGGCACCACCCCTGAGGTCCATAGCATCTTCCTGGAGGGCCACACTTTCCTGGTGAGGAACCACAGACAGGCCTCTCTGGAGATCTCTCCCATCACCTTCCTGACTGCTCAGACTCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTTTGCCATATTAGCAGCCACCAGCATGATGGGATGGAGGCCTATGTGAAGGTGGATAGCTGCCCTGAGGAGCCTCAGCTGAGGATGAAGAACAATGAGGAGGCTGAAGACTATGATGATGACCTGACTGATTCTGAGATGGATGTGGTGAGGTTTGATGATGACAATAGCCCCAGCTTCATTCAGATCAGGTCTGTGGCCAAGAAACACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAAGAGGACTGGGACTATGCTCCCCTGGTGCTGGCCCCTGATGATAGGTCTTATAAGAGCCAGTACCTGAACAATGGGCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTGAGGTTCATGGCCTACACTGATGAAACCTTCAAAACCAGGGAGGCCATTCAGCATGAGTCTGGCATCCTGGGCCCTCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTATCCTCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAAGACTTCCCCATCCTGCCTGGGGAGATCTTTAAGTATAAGTGGACTGTGACTGTGGAGGATGGCCCTACCAAGTCTGACCCCAGGTGTCTGACCAGGTACTATTCTAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGGCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTTTCTGTGTTTGATGAGAATAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTTCTGCCCAATCCTGCTGGGGTGCAGCTGGAGGATCCTGAGTTCCAGGCCAGCAATATCATGCATAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATGGGGCCCAGACTGACTTTCTGTCTGTGTTCTTTTCTGGCTATACCTTCAAGCACAAGATGGTGTATGAGGATACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAATCCTGGGCTGTGGATCCTGGGGTGCCACAACTCTGATTTTAGGAACAGGGGGATGACTGCCCTGCTGAAGGTGTCTAGCTGTGATAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATTTCTGCTTATCTGCTGTCTAAGAATAATGCCATTGAGCCCAGAAGCTTCAGCCAGAATCCCCCTGTGCTGAAGAGACATCAGAGGGAGATCACCAGAACTACCCTGCAGTCTGATCAGGAGGAGATTGACTATGATGACACTATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTATGATGAGGATGAGAATCAGTCTCCCAGGAGCTTTCAGAAGAAGACCAGACATTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCTCTAGCCCTCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAATTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGGGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGGCCTTATATCAGGGCTGAGGTGGAGGATAATATTATGGTGACTTTCAGGAACCAGGCCAGCAGGCCCTACTCTTTCTATAGCAGCCTGATCTCTTATGAGGAGGATCAGAGGCAGGGGGCTGAGCCTAGGAAGAACTTTGTGAAGCCCAATGAGACTAAGACCTACTTCTGGAAGGTCCAGCACCACATGGCCCCTACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTATTTCTCTGATGTGGATCTGGAGAAGGATGTCCATTCTGGGCTGATTGGCCCCCTGCTGGTGTGCCACACTAACACTCTGAATCCTGCCCATGGCAGGCAGGTGACTGTCCAGGAGTTTGCCCTGTTCTTCACTATCTTTGATGAGACCAAGAGCTGGTACTTTACTGAGAACATGGAGAGGAACTGCAGAGCTCCTTGCAATATTCAGATGGAGGACCCCACCTTCAAGGAGAATTACAGGTTCCATGCCATTAATGGGTACATCATGGACACCCTGCCTGGCCTGGTGATGGCTCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCTCTAATGAGAATATCCACAGCATCCACTTCTCTGGGCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCTCTGTATAATCTGTACCCTGGGGTGTTTGAAACTGTGGAGATGCTGCCCTCTAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTATTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATTCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCTCTCTGTACATCTCTCAGTTCATCATCATGTACTCTCTGGATGGGAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGGAATGTGGACTCTTCTGGCATCAAGCACAACATCTTCAATCCCCCCATCATTGCTAGGTATATTAGGCTGCATCCCACCCACTACAGCATCAGGTCTACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACTCTTGCAGCATGCCCCTGGGCATGGAGTCTAAGGCCATCTCTGATGCCCAGATTACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCTCTAAGGCCAGGCTGCATCTGCAGGGGAGGAGCAATGCCTGGAGGCCTCAGGTGAACAACCCCAAGGAGTGGCTGCAGGTGGATTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTCAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACTCTGTTCTTTCAGAATGGGAAGGTGAAGGTGTTTCAGGGCAATCAGGACTCTTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATCCACCCCCAGTCTTGGGTGCATCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCTCAGGATCTGTACTGA B domain-deleted 4374WO20170   9 382ATGCAGATCGAGCTCTCCACCTGCTTCTTTCTGTGCCTGTTGAGATTCTGCTTCAGCGCCACCAGGAGATACTACCTGGG(BDD) hFVIII with 74526A1GGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTSQ sequencel IIGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATCCACCCGTCCTTAAGCGCCATCAGCGCGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCATCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA Endogenous 7056NG_  70 383ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGThFVIII cDNA 011403.1GCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTGCCAAAATCTTTTCCATTCAACACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTTTCAACATCGCTAAGCCAAGGCCACCCTGGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTCAGTCTTCATGCTGTTGGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGGGAGAAAGAAGATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCACTGTGCCTTACCTACTCATATCTTTCTCATGTGGACCTGGTAAAAGACTTGAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAAGGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAATTTATACTACTTTTTGCTGTATTTGATGAAGGGAAAAGTTGGCACTCAGAAACAAAGAACTCCTTGATGCAGGATAGGGATGCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGGTTATGTAAACAGGTCTCTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATTGGCATGTGATTGGAATGGGCACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTCACACATTTCTTGTGAGGAACCATCGCCAGGCGTCCTTGGAAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTCTTGATGGACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGCATGGAAGCTTATGTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAAGAAGCGGAAGACTATGATGATGATCTTACTGATTCTGAAATGGATGTGGTCAGGTTTGATGATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCCAAGAAGCATCCTAAAACTTGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCCCCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAAGTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCTATTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTGTTGATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCGTCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTTCCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAATCAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAGAACCGAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAGTTCCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGTTTGCAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGCATTGGAGCACAGACTGACTTCCTTTCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACACACTCACCCTATTCCCATTCTCAGGAGAAACTGTCTTCATGTCGATGGAAAACCCAGGTCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAACAGAGGCATGACCGCCTTACTGAAGGTTTCTAGTTGTGACAAGAACACTGGTGATTATTACGAGGACAGTTATGAAGATATTTCAGCATACTTGCTGAGTAAAAACAATGCCATTGAACCAAGAAGCTTCTCCCAGAATTCAAGACACCCTAGCACTAGGCAAAAGCAATTTAATGCCACCACAATTCCAGAAAATGACATAGAGAAGACTGACCCTTGGTTTGCACACAGAACACCTATGCCTAAAATACAAAATGTCTCCTCTAGTGATTTGTTGATGCTCTTGCGACAGAGTCCTACTCCACATGGGCTATCCTTATCTGATCTCCAAGAAGCCAAATATGAGACTTTTTCTGATGATCCATCACCTGGAGCAATAGACAGTAATAACAGCCTGTCTGAAATGACACACTTCAGGCCACAGCTCCATCACAGTGGGGACATGGTATTTACCCCTGAGTCAGGCCTCCAATTAAGATTAAATGAGAAACTGGGGACAACTGCAGCAACAGAGTTGAAGAAACTTGATTTCAAAGTTTCTAGTACATCAAATAATCTGATTTCAACAATTCCATCAGACAATTTGGCAGCAGGTACTGATAATACAAGTTCCTTAGGACCCCCAAGTATGCCAGTTCATTATGATAGTCAATTAGATACCACTCTATTTGGCAAAAAGTCATCTCCCCTTACTGAGTCTGGTGGACCTCTGAGCTTGAGTGAAGAAAATAATGATTCAAAGTTGTTAGAATCAGGTTTAATGAATAGCCAAGAAAGTTCATGGGGAAAAAATGTATCGTCAACAGAGAGTGGTAGGTTATTTAAAGGGAAAAGAGCTCATGGACCTGCTTTGTTGACTAAAGATAATGCCTTATTCAAAGTTAGCATCTCTTTGTTAAAGACAAACAAAACTTCCAATAATTCAGCAACTAATAGAAAGACTCACATTGATGGCCCATCATTATTAATTGAGAATAGTCCATCAGTCTGGCAAAATATATTAGAAAGTGACACTGAGTTTAAAAAAGTGACACCTTTGATTCATGACAGAATGCTTATGGACAAAAATGCTACAGCTTTGAGGCTAAATCATATGTCAAATAAAACTACTTCATCAAAAAACATGGAAATGGTCCAACAGAAAAAAGAGGGCCCCATTCCACCAGATGCACAAAATCCAGATATGTCGTTCTTTAAGATGCTATTCTTGCCAGAATCAGCAAGGTGGATACAAAGGACTCATGGAAAGAACTCTCTGAACTCTGGGCAAGGCCCCAGTCCAAAGCAATTAGTATCCTTAGGACCAGAAAAATCTGTGGAAGGTCAGAATTTCTTGTCTGAGAAAAACAAAGTGGTAGTAGGAAAGGGTGAATTTACAAAGGACGTAGGACTCAAAGAGATGGTTTTTCCAAGCAGCAGAAACCTATTTCTTACTAACTTGGATAATTTACATGAAAATAATACACACAATCAAGAAAAAAAAATTCAGGAAGAAATAGAAAAGAAGGAAACATTAATCCAAGAGAATGTAGTTTTGCCTCAGATACATACAGTGACTGGCACTAAGAATTTCATGAAGAACCTTTTCTTACTGAGCACTAGGCAAAATGTAGAAGGTTCATATGACGGGGCATATGCTCCAGTACTTCAAGATTTTAGGTCATTAAATGATTCAACAAATAGAACAAAGAAACACACAGCTCATTTCTCAAAAAAAGGGGAGGAAGAAAACTTGGAAGGCTGGGAAATCAAACCAAGCAAATTGTAGAGAAATATGCATGCACCACAAGGATATCTCCTAATACAAGCCAGCAGAATTTTGTCACGCAACGTAGTAAGAGAGCTTTGAAACAATTCAGACTCCCACTAGAAGAAACAGAACTTGAAAAAAGGATAATTGTGGATGACACCTCAACCCAGTGGTCCAAAAACATGAAACATTTGACCCCGAGCACCCTCACACAGATAGACTACAATGAGAAGGAGAAAGGGGCCATTACTCAGTCTCCCTTATCAGATTGCCTTACGAGGAGTCATAGCATCCCTCAAGCAAATAGATCTCCATTACCCATTGCAAAGGTATCATCATTTCCATCTATTAGACCTATATATCTGACCAGGGTCCTATTCCAAGACAACTCTTCTCATCTTCCAGCAGCATCTTATAGAAAGAAAGATTCTGGGGTCCAAGAAAGCAGTCATTTCTTACAAGGAGCCAAAAAAAATAACCTTTCTTTAGCCATTCTAACCTTGGAGATGACTGGTGATCAAAGAGAGGTTGGCTCCCTGGGGACAAGTGCCACAAATTCAGTCACATACAAGAAAGTTGAGAACACTGTTCTCCCGAAACCAGACTTGCCCAAAACATCTGGCAAAGTTGAATTGCTTCCAAAAGTTCACATTTATCAGAAGGACCTATTCCCTACGGAAACTAGCAATGGGTCTCCTGGCCATCTGGATCTCGTGGAAGGGAGCCTTCTTCAGGGAACAGAGGGAGCGATTAAGTGGAATGAAGCAAACAGACCTGGAAAAGTTCCCTTTCTGAGAGTAGCAACAGAAAGCTCTGCAAAGACTCCCTCCAAGCTATTGGATCCTCTTGCTGGGATAACCACTATGGTACTCAGATACCAAAAGAAGAGTGGAAATCCCAAGAGAAGTCACCAGAAAAAACAGCTTTTAAGAAAAAGGATACCATTTTGTCCCTGAACGCTTGTGAAAGCAATCATGCAATAGCAGCAATAAATGAGGGACAAAATAAGCCCGAAATAGAAGTCACCTGGGCAAAGCAAGGTAGGACTGAAAGGCTGTGCTCTCAAAACCCACCAGTCTTGAAACGCCATCAACGGGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGACTATGATGATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACACGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGGATTATGGGATGAGTAGCTCCCCACATGTTCTAAGAAACAGGGCTCAGAGTGGCAGTGTCCCTCAGTTCAAGAAAGTTGTTTTCCAGGAATTTACTGATGGCTCCTTTACTCAGCCCTTATACCGTGGAGAACTAAATGAACATTTGGGACTCCTGGGGCCATATATAAGAGCAGAAGTTGAAGATAATATCATGGTAACTTTCAGAAATCAGGCCTCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGGAAGATCAGAGGCAAGGAGCAGAACCTAGAAAAAACTTTGTCAAGCCTAATGAAACCAAAACTTACTTTTGGAAAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGGGCTTATTTCTCTGATGTTGACCTGGAAAAAGATGTGCACTCAGGCCTGATTGGACCCCTTCTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGGAATTTGCTCTGTTTTTCACCATCTTTGATGAGACCAAAAGCTGGTACTTCACTGAAAATATGGAAAGAAACTGCAGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAATTATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGGCTTAGTAATGGCTCAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCTATTCATTTCAGTGGACATGTGTTCACTGTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTATCCAGGTGTTTTTGAGACAGTGGAAATGTTACCATCCAAAGCTGGAATTTGGCGGGTGGAATGCCTTATTGGCGAGCATCTACATGCTGGGATGAGCACACTTTTTCTGGTGTACAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTTCAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTGTTGGCACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGCCTCTACATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATAAAACACAATATTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCATTATAGCATTCGCAGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGCATGCCATTGGGAATGGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTACTTTACCAATATGTTTGCCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGGAGTAATGCCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGGAGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTTCAGAATGGCAAAGTAAAGGTTTTTCAGGGAAATCAAGACTCCTTCACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGACTCGCTACCTTCGAATTCACCCCCAGAGTTGGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTCTACTGAB domain-deleted 4425 https://   3 384ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGG(BDD) hEVIII with www.ncbi.GGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTSQ sequence, 6 nlm.nih.GCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACAN-linked gov/TTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACglycosylation pubmed/CCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAsites, and furin 23426947GTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCcleavage siteAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGT(SFSQNATNVSNNSNTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGASNDSNVSPPVLKRHQR)CCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATGCCACTAATGTGTCTAACAACAGCAACACCAGCAATGACAGCAATGTGTCTCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAG-MGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA B domain-deleted 385MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRP(BDD) hFVIII withPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGSQ sequence, 6PMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWN-linkedPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHIglycosylationSSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDsites, and furin WDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPcleavageHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRsite-amino acid GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQsequenceTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNATNVSNNSNTSNDSNVSPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPANGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEELISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY-B domain-deleted 5055 US  92 386atgcaaatagagctctccacctgcttctttctgtgattttgcgattctgctttagtgccaccagaagatactacctgggtgcagtggaact(BDD) hFVIII with 9,050,318gtcatgggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacaccSQ sequence B2tcagtcgtgtacaaaaagactctgtttgtagaattcacggatcacatttcaacatcgctaagccaaggccaccctggatgggtctgctaggfused to FctcctaccatccaggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtaregiontcctactggaaagcttctgagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcctcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagtcaatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattggaatgggcaccactcctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaaacactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcttatgtcaaagtagacagctgtccagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttgatgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcggattggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggggaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaagatgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatggagagagatctagcttcaggactcattggccctctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaagaggaatgtcatcctgttttctgtatttgatgagaaccgaagctggtacctcacagagaatatacaacgctttctccccaatccagctggagtgcagcttgaggatccagagttccaagcctccaacatcatgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggtacattctaagcattggagcacagactgacttcctttctgtcttcttctctggatataccttcaaacacaaaatggtctatgaagacacactcaccctattcccattctcaggagaaactgtcttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgaccgccttactgaaggtttctagttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatacttgctgagtaaaaacaatgccattgaaccaagaagcttctctcaaaacccaccagtcttgaaacgccatcaacgggaaataactcgtactactcttcagtcagatcaagaggaaattgactatgatgataccatatcagttgaaatgaagaaggaagattttgacatttatgatgaggatgaaaatcagagcccccgcagctttcaaaagaaaacacgacactattttattgctgcagtggagaggctctgggattatgggatgagtagctccccacatgttctaagaaacagggctcagagtggcagtgtccctcagttcaagaaagttgttttccaggaatttactgatggctcctttactcagcccttataccgtggagaactaaatgaacatttgggactcctggggccatatataagagcagaagttgaagataatatcatggtaactttcagaaatcaggcctctcgtccctattccttctattctagccttatttcttatgaggaagatcagaggcaaggagcagaacctagaaaaaactttgtcaagcctaatgaaaccaaaacttacttttggaaagtgcaacatcatatggcacccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctggaaaaagatgtgcactcaggcctgattggaccccttctggtctgccacactaacacactgaaccctgctcatgggagacaagtgacagtacaggaatttgctctgtttttcaccatctttgatgagaccaaaagctggtacttcactgaaaatatggaaagaaactgcagggctccctgcaatatccagatggaagatcccacttttaaagagaattatcgcttccatgcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatcaaaggattcgatggtatctgctcagcatgggcagcaatgaaaacatccattctattcatttcagtggacatgtgttcactgtacgaaaaaaagaggagtataaaatggcactgtacaatctctatccaggtgtttttgagacagtggaaatgttaccatccaaagctggaatttggcgggtggaatgccttattggcgagcatctacatgctgggatgagcacactttttctggtgtacagcaataagtgtcagactcccctgggaatggcttctggacacattagagattttcagattacagcttcaggacaatatggacagtgggccccaaagctggccagacttcattattccggatcaatcaatgcctggagcaccaaggagcccttttcttggatcaaggtggatctgttggcaccaatgattattcacggcatcaagacccagggtgcccgtcagaagttctccagcctctacatctctcagtttatcatcatgtatagtcttgatgggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttctttggcaatgtggattcatctgggataaaacacaatatttttaaccctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgcagcactcttcgcatggagttgatgggctgtgatttaaatagttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattactgcttcatcctactttaccaatatgtttgccacctggtctccttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggtgaataatccaaaagagtggctgcaagtggacttccagaagacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttaccagcatgtatgtgaaggagttcctcatctccagcagtcaagatggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcagggaaatcaagactccttcacacctgtggtgaactctctagacccaccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccagattgccctgaggatggaggttctgggctgcgaggcacaggacctctacgacaaaactcacacatgcccaccgtgcccagctccagaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaB domain-deleted 4374 211 387ATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGG(BDD)CGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGThEVIII with SQGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATAsequenceTCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCAC(codop)CCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCFTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAG Jcat optimized B- 4980350 388ATGCAGATCGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGCGCTTCTGCTTCAGCGCCACCCGCCGCTACTACCTGGGdomainCGCCGTGGAGCTGAGCTGGGACTACATGCAGAGCGACCTGGGCGAGCTGCCCGTGGACGCCCGCTTCCCCCCCCGCGTtruncated (206GCCCAAGAGCTTCCCCTTCAACACCAGCGTGGTGTACAAGAAGACCCTGTTCGTGGAGTTCACCGACCACCTGTTCAACAamino acidsTCGCCAAGCCCCGCCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCCGAGGTGTACGACACCGTGGTGATCACremaining) HumanCCTGAAGAACATGGCCAGCCACCCCGTGAGCCTGCACGCCGTGGGCGTGAGCTACTGGAAGGCCAGCGAGGGCGCCGFactor VIIIAGTACGACGACCAGACCAGCCAGCGCGAGAAGGAGGACGACAAGGTGTTCCCCGGCGGCAGCCACACCTACGTGTGGORF (BDD-206)CAGGTGCTGAAGGAGAACGGCCCCATGGCCAGCGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCACGTGGACCTGGTGAAGGACCTGAACAGCGGCCTGATCGGCGCCCTGCTGGTGTGCCGCGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACCAAGAACAGCCTGATGCAGGACCGCGACGCCGCCAGCGCCCGCGCCTGGCCCAAGATGCACACCGTGAACGGCTACGTGAACCGCAGCCTGCCCGGCCTGATCGGCTGCCACCGCAAGAGCGTGTACTGGCACGTGATCGGCATGGGCACCACCCCCGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTGCGCAACCACCGCCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACCGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCACGACGGCATGGAGGCCTACGTGAAGGTGGACAGCTGCCCCGAGGAGCCCCAGCTGCGCATGAAGAACAACGAGGAGGCCGAGGACTACGACGACGACCTGACCGACAGCGAGATGGACGTGGTGCGCTTCGACGACGACAACAGCCCCAGCTTCATCCAGATCCGCAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATCGCCGCCGAGGAGGAGGACTGGGACTACGCCCCCCTGGTGCTGGCCCCCGACGACCGCAGCTACAAGAGCCAGTACCTGAACAACGGCCCCCAGCGCATCGGCCGCAAGTACAAGAAGGTGCGCTTCATGGCCTACACCGACGAGACCTTCAAGACCCGCGAGGCCATCCAGCACGAGAGCGGCATCCTGGGCCCCCTGCTGTACGGCGAGGTGGGCGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCCGCCCCTACAACATCTACCCCCACGGCATCACCGACGTGCGCCCCCTGTACAGCCGCCGCCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCCATCCTGCCCGGCGAGATCTTCAAGTACAAGTGGACCGTGACCGTGGAGGACGGCCCCACCAAGAGCGACCCCCGCTGCCTGACCCGCTACTACAGCAGCTTCGTGAACATGGAGCGCGACCTGGCCAGCGGCCTGATCGGCCCCCTGCTGATCTGCTACAAGGAGAGCGTGGACCAGCGCGGCAACCAGATCATGAGCGACAAGCGCAACGTGATCCTGTTCAGCGTGTTCGACGAGAACCGCAGCTGGTACCTGACCGAGAACATCCAGCGCTTCCTGCCCAACCCCGCCGGCGTGCAGCTGGAGGACCCCGAGTTCCAGGCCAGCAACATCATGCACAGCATCAACGGCTACGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACATCCTGAGCATCGGCGCCCAGACCGACTTCCTGAGCGTGTTCTTCAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGACACCCTGACCCTGTTCCCCTTCAGCGGCGAGACCGTGTTCATGAGCATGGAGAACCCCGGCCTGTGGATCCTGGGCTGCCACAACAGCGACTTCCGCAACCGCGGCATGACCGCCCTGCTGAAGGTGAGCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTACGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAACGCCATCGAGCCCCGCAGCTTCAGCCAGAACAGCCGCCACCCCAGCACCCGCCAGAAGCAGTTCAACGCCACCACCATCCCCGAGAACGACATCGAGAAGACCGACCCCTGGTTCGCCCACCGCACCCCCATGCCCAAGATCCAGAACGTGAGCAGCAGCGACCTGCTGATGCTGCTGCGCCAGAGCCCCACCCCCCACGGCCTGAGCCTGAGCGACCTGCAAGAGGCCAAGTACGAGACCTTCAGCGACGACCCCAGCCCCGGCGCCATCGACAGCAACAACAGCCTGAGCGAGATGACCCACTTCCGCCCCCAGCTGCACCACAGCGGCGACATGGTGTTCACCCCCGAGAGCGGCCTGCAGCTGCGCCTGAACGAGAAGCTGGGCACCACCGCCGCCACCGAGCTGAAGAAGCTGGACTTCAAGGTGAGCAGCACCAGCAACAACCTGATCAGCACCATCCCCAGCGACAACCTGGCCGCCGGCACCGACAACACCAGCAGCCTGGGCCCCCCCAGCATGCCCGTGCACTACGACAGCCAGCTGGACACCACCCTGTTCGGCAAGAAGAGCAGCCCCCTGACCGAGAGCGGCGGCCCCCTGAGCCTGAGCGAGGAGAACAACGACAGCAAGCTGCCCCCCGTGCTGAAGCGCCACCAGCGCGAGATCACCCGCACCACCCTGCAGAGCGACCAGGAGGAGATCGACTACGACGACACCATCAGCGTGGAGATGAAGAAGGAGGACTTCGACATCTACGACGAGGACGAGAACCAGAGCCCCCGCAGCTTCCAGAAGAAGACCCGCCACTACTTCATCGCCGCCGTGGAGCGCCTGTGGGACTACGGCATGAGCAGCAGCCCCCACGTGCTGCGCAACCGCGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCCCTGTACCGCGGCGAGCTGAACGAGCACCTGGGCCTGCTGGGCCCCTACATCCGCGCCGAGGTGGAGGACAACATCATGGTGACCTTCCGCAACCAGGCCAGCCGCCCCTACAGCTTCTACAGCAGCCTGATCAGCTACGAGGAGGACCAGCGCCAGGGCGCCGAGCCCCGCAAGAACTTCGTGAAGCCCAACGAGACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCAGCGACGTGGACCTGGAGAAGGACGTGCACAGCGGCCTGATCGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCCGCCCACGGCCGCCAGGTGACCGTGCAGGAGTTCGCCCTGTTCTTCACCATCTTCGACGAGACCAAGAGCTGGTACTTCACCGAGAACATGGAGCGCAACTGCCGCGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACCGCTTCCACGCCATCAACGGCTACATCATGGACACCCTGCCCGGCCTGGTGATGGCCCAGGACCAGCGCATCCGCTGGTACCTGCTGAGCATGGGCAGCAACGAGAACATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGCAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCCGGCGTGTTCGAGACCGTGGAGATGCTGCCCAGCAAGGCCGGCATCTGGCGCGTGGAGTGCCTGATCGGCGAGCACCTGCACGCCGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCAGCGGCCACATCCGCGACTTCCAGATCACCGCCAGCGGCCAGTACGGCCAGTGGGCCCCCAAGCTGGCCCGCCTGCACTACAGCGGCAGCATCAACGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCACGGCATCAAGACCCAGGGCGCCCGCCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACCGCGGCAACAGCACCGGCACCCTGATGGTGTTCTTCGGCAACGTGGACAGCAGCGGCATCAAGCACAACATCTTCAACCCCCCCATCATCGCCCGCTACATCCGCCTGCACCCCACCCACTACAGCATCCGCAGCACCCTGCGCATGGAGCTGATGGGCTGCGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCAGCGACGCCCAGATCACCGCCAGCAGCTACTTCACCAACATGTTCGCCACCTGGAGCCCCAGCAAGGCCCGCCTGCACCTGCAAGGCCGCAGCAACGCCTGGCGCCCCCAGGTGAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACCGGCGTGACCACCCAGGGCGTGAAGAGCCTGCTGACCAGCATGTACGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCCGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCCGCTACCTGCGCATCCACCCCCAGAGCTGGGTGCACCAGATCGCCCTGCGCATGGAGGTGCTGGGCTGCGAGGCCCAGGACCTGTACTAA Jcat optimized 7056 467389ATGCAGATCGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGCGCTTCTGCTTCAGCGCCACCCGCCGCTACTACCTGGGWild typeCGCCGTGGAGCTGAGCTGGGACTACATGCAGAGCGACCTGGGCGAGCTGCCCGTGGACGCCCGCTTCCCCCCCCGCGTHuman FactorGCCCAAGAGCTTCCCCTTCAACACCAGCGTGGTGTACAAGAAGACCCTGTTCGTGGAGTTCACCGACCACCTGTTCAACAVIII ORFTCGCCAAGCCCCGCCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCCGAGGTGTACGACACCGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCCGTGAGCCTGCACGCCGTGGGCGTGAGCTACTGGAAGGCCAGCGAGGGCGCCGAGTACGACGACCAGACCAGCCAGCGCGAGAAGGAGGACGACAAGGTGTTCCCCGGCGGCAGCCACACCTACGTGTGGCAGGTGCTGAAGGAGAACGGCCCCATGGCCAGCGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCACGTGGACCTGGTGAAGGACCTGAACAGCGGCCTGATCGGCGCCCTGCTGGTGTGCCGCGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACCAAGAACAGCCTGATGCAGGACCGCGACGCCGCCAGCGCCCGCGCCTGGCCCAAGATGCACACCGTGAACGGCTACGTGAACCGCAGCCTGCCCGGCCTGATCGGCTGCCACCGCAAGAGCGTGTACTGGCACGTGATCGGCATGGGCACCACCCCCGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTGCGCAACCACCGCCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACCGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCACGACGGCATGGAGGCCTACGTGAAGGTGGACAGCTGCCCCGAGGAGCCCCAGCTGCGCATGAAGAACAACGAGGAGGCCGAGGACTACGACGACGACCTGACCGACAGCGAGATGGACGTGGTGCGCTTCGACGACGACAACAGCCCCAGCTTCATCCAGATCCGCAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATCGCCGCCGAGGAGGAGGACTGGGACTACGCCCCCCTGGTGCTGGCCCCCGACGACCGCAGCTACAAGAGCCAGTACCTGAACAACGGCCCCCAGCGCATCGGCCGCAAGTACAAGAAGGTGCGCTTCATGGCCTACACCGACGAGACCTTCAAGACCCGCGAGGCCATCCAGCACGAGAGCGGCATCCTGGGCCCCCTGCTGTACGGCGAGGTGGGCGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCCGCCCCTACAACATCTACCCCCACGGCATCACCGACGTGCGCCCCCTGTACAGCCGCCGCCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCCATCCTGCCCGGCGAGATCTTCAAGTACAAGTGGACCGTGACCGTGGAGGACGGCCCCACCAAGAGCGACCCCCGCTGCCTGACCCGCTACTACAGCAGCTTCGTGAACATGGAGCGCGACCTGGCCAGCGGCCTGATCGGCCCCCTGCTGATCTGCTACAAGGAGAGCGTGGACCAGCGCGGCAACCAGATCATGAGCGACAAGCGCAACGTGATCCTGTTCAGCGTGTTCGACGAGAACCGCAGCTGGTACCTGACCGAGAACATCCAGCGCTTCCTGCCCAACCCCGCCGGCGTGCAGCTGGAGGACCCCGAGTTCCAGGCCAGCAACATCATGCACAGCATCAACGGCTACGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACATCCTGAGCATCGGCGCCCAGACCGACTTCCTGAGCGTGTTCTTCAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGACACCCTGACCCTGTTCCCCTTCAGCGGCGAGACCGTGTTCATGAGCATGGAGAACCCCGGCCTGTGGATCCTGGGCTGCCACAACAGCGACTTCCGCAACCGCGGCATGACCGCCCTGCTGAAGGTGAGCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTACGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAACGCCATCGAGCCCCGCAGCTTCAGCCAGAACAGCCGCCACCCCAGCACCCGCCAGAAGCAGTTCAACGCCACCACCATCCCCGAGAACGACATCGAGAAGACCGACCCCTGGTTCGCCCACCGCACCCCCATGCCCAAGATCCAGAACGTGAGCAGCAGCGACCTGCTGATGCTGCTGCGCCAGAGCCCCACCCCCCACGGCCTGAGCCTGAGCGACCTGCAAGAGGCCAAGTACGAGACCTTCAGCGACGACCCCAGCCCCGGCGCCATCGACAGCAACAACAGCCTGAGCGAGATGACCCACTTCCGCCCCCAGCTGCACCACAGCGGCGACATGGTGTTCACCCCCGAGAGCGGCCTGCAGCTGCGCCTGAACGAGAAGCTGGGCACCACCGCCGCCACCGAGCTGAAGAAGCTGGACTTCAAGGTGAGCAGCACCAGCAACAACCTGATCAGCACCATCCCCAGCGACAACCTGGCCGCCGGCACCGACAACACCAGCAGCCTGGGCCCCCCCAGCATGCCCGTGCACTACGACAGCCAGCTGGACACCACCCTGTTCGGCAAGAAGAGCAGCCCCCTGACCGAGAGCGGCGGCCCCCTGAGCCTGAGCGAGGAGAACAACGACAGCAAGCTGCTGGAGAGCGGCCTGATGAACAGCCAGGAGAGCAGCTGGGGCAAGAACGTGAGCAGCACCGAGAGCGGCAGGCTGTTCAAGGGCAAGCGCGCCCACGGCCCCGCCCTGCTGACCAAGGACAACGCCCTGTTCAAGGTGAGCATCAGCCTGCTGAAGACCAACAAGACCAGCAACAACAGCGCCACCAACCGCAAGACCCACATCGACGGCCCCAGCCTGCTGATCGAGAACAGCCCCAGCGTGTGGCAGAACATCCTGGAGAGCGACACCGAGTTCAAGAAGGTGACCCCCCTGATCCACGACCGCATGCTGATGGACAAGAACGCCACCGCCCTGCGCCTGAACCACATGAGCAACAAGACCACCAGCAGCAAGAACATGGAGATGGTGCAGCAGAAGAAGGAGGGCCCCATCCCCCCCGACGCCCAGAACCCCGACATGAGCTTCTTCAAGATGCTGTTCCTGCCCGAGAGCGCCCGCTGGATCCAGCGCACCCACGGCAAGAACAGCCTGAACAGCGGCCAGGGCCCCAGCCCCAAGCAGCTGGTGAGCCTGGGCCCCGAGAAGAGCGTGGAGGGCCAGAACTTCCTGAGCGAGAAGAACAAGGTGGTGGTGGGCAAGGGCGAGTTCACCAAGGACGTGGGCCTGAAGGAGATGGTGTTCCCCAGCAGCCGCAACCTGTTCCTGACCAACCTGGACAACCTGCACGAGAACAACACCCACAACCAGGAGAAGAAGATCCAGGAGGAGATCGAGAAGAAGGAGACCCTGATCCAGGAGAACGTGGTGCTGCCCCAGATCCACACCGTGACCGGCACCAAGAACTTCATGAAGAACCTGTTCCTGCTGAGCACCCGCCAGAACGTGGAGGGCAGCTACGACGGCGCCTACGCCCCCGTGCTGCAGGACTTCCGCAGCCTGAACGACAGCACCAACCGCACCAAGAAGCACACCGCCCACTTCAGCAAGAAGGGCGAGGAGGAGAACCTGGAGGGCCTGGGCAACCAGACCAAGCAGATCGTGGAGAAGTACGCCTGCACCACCCGCATCAGCCCCAACACCAGCCAGCAGAACTTCGTGACCCAGCGCAGCAAGCGCGCCCTGAAGCAGTTCCGCCTGCCCCTGGAGGAGACCGAGCTGGAGAAGCGCATCATCGTGGACGACACCAGCACCCAGTGGAGCAAGAACATGAAGCACCTGACCCCCAGCACCCTGACCCAGATCGACTACAACGAGAAGGAGAAGGGCGCCATCACCCAGAGCCCCCTGAGCGACTGCCTGACCCGCAGCCACAGCATCCCCCAGGCCAACCGCAGCCCCCTGCCCATCGCCAAGGTGAGCAGCTTCCCCAGCATCCGCCCCATCTACCTGACCCGCGTGCTGTTCCAGGACAACAGCAGCCACCTGCCCGCCGCCAGCTACCGCAAGAAGGACAGCGGCGTGCAGGAGAGCAGCCACTTCCTGCAAGGCGCCAAGAAGAACAACCTGAGCCTGGCCATCCTGACCCTGGAGATGACCGGCGACCAGCGCGAGGTGGGCAGCCTGGGCACCAGCGCCACCAACAGCGTGACCTACAAGAAGGTGGAGAACACCGTGCTGCCCAAGCCCGACCTGCCCAAGACCAGCGGCAAGGTGGAGCTGCTGCCCAAGGTGCACATCTACCAGAAGGACCTGTTCCCCACCGAGACCAGCAACGGCAGCCCCGGCCACCTGGACCTGGTGGAGGGCAGCCTGCTGCAGGGCACCGAGGGCGCCATCAAGTGGAACGAGGCCAACCGCCCCGGCAAGGTGCCCTTCCTGCGCGTGGCCACCGAGAGCAGCGCCAAGACCCCCAGCAAGCTGCTGGACCCCCTGGCCTGGGACAACCACTACGGCACCCAGATCCCCAAGGAGGAGTGGAAGAGCCAGGAGAAGAGCCCCGAGAAGACCGCCTTCAAGAAGAAGGACACCATCCTGAGCCTGAACGCCTGCGAGAGCAACCACGCCATCGCCGCCATCAACGAGGGCCAGAACAAGCCCGAGATCGAGGTGACCTGGGCCAAGCAGGGCCGCACCGAGCGCCTGTGCAGCCAGAACCCCCCCGTGCTGAAGCGCCACCAGCGCGAGATCACCCGCACCACCCTGCAGAGCGACCAGGAGGAGATCGACTACGACGACACCATCAGCGTGGAGATGAAGAAGGAGGACTTCGACATCTACGACGAGGACGAGAACCAGAGCCCCCGCAGCTTCCAGAAGAAGACCCGCCACTACTTCATCGCCGCCGTGGAGCGCCTGTGGGACTACGGCATGAGCAGCAGCCCCCACGTGCTGCGCAACCGCGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCCCTGTACCGCGGCGAGCTGAACGAGCACCTGGGCCTGCTGGGCCCCTACATCCGCGCCGAGGTGGAGGACAACATCATGGTGACCTTCCGCAACCAGGCCAGCCGCCCCTACAGCTTCTACAGCAGCCTGATCAGCTACGAGGAGGACCAGCGCCAGGGCGCCGAGCCCCGCAAGAACTTCGTGAAGCCCAACGAGACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCAGCGACGTGGACCTGGAGAAGGACGTGCACAGCGGCCTGATCGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCCGCCCACGGCCGCCAGGTGACCGTGCAGGAGTTCGCCCTGTTCTTCACCATCTTCGACGAGACCAAGAGCTGGTACTTCACCGAGAACATGGAGCGCAACTGCCGCGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACCGCTTCCACGCCATCAACGGCTACATCATGGACACCCTGCCCGGCCTGGTGATGGCCCAGGACCAGCGCATCCGCTGGTACCTGCTGAGCATGGGCAGCAACGAGAACATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGCAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCCGGCGTGTTCGAGACCGTGGAGATGCTGCCCAGCAAGGCCGGCATCTGGCGCGTGGAGTGCCTGATCGGCGAGCACCTGCACGCCGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCAGCGGCCACATCCGCGACTTCCAGATCACCGCCAGCGGCCAGTACGGCCAGTGGGCCCCCAAGCTGGCCCGCCTGCACTACAGCGGCAGCATCAACGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCACGGCATCAAGACCCAGGGCGCCCGCCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACCGCGGCAACAGCACCGGCACCCTGATGGTGTTCTTCGGCAACGTGGACAGCAGCGGCATCAAGCACAACATCTTCAACCCCCCCATCATCGCCCGCTACATCCGCCTGCACCCCACCCACTACAGCATCCGCAGCACCCTGCGCATGGAGCTGATGGGCTGCGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCAGCGACGCCCAGATCACCGCCAGCAGCTACTTCACCAACATGTTCGCCACCTGGAGCCCCAGCAAGGCCCGCCTGCACCTGCAAGGCCGCAGCAACGCCTGGCGCCCCCAGGTGAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACCGGCGTGACCACCCAGGGCGTGAAGAGCCTGCTGACCAGCATGTACGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCCGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCCGCTACCTGCGCATCCACCCCCAGAGCTGGGTGCACCAGATCGCCCTGCGCATGGAGGTGCTGGGCTGCGAGGCCCAGGACCTGTACTAA Jcat optimized B- 4374 307 390ATGCAGATCGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGCGCTTCTGCTTCAGCGCCACCCGCCGCTACTACCTGGGdomainCGCCGTGGAGCTGAGCTGGGACTACATGCAGAGCGACCTGGGCGAGCTGCCCGTGGACGCCCGCTTCCCCCCCCGCGTdeleted HumanGCCCAAGAGCTTCCCCTTCAACACCAGCGTGGTGTACAAGAAGACCCTGTTCGTGGAGTTCACCGACCACCTGTTCAACAFactor VIIITCGCCAAGCCCCGCCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCCGAGGTGTACGACACCGTGGTGATCACORFCCTGAAGAACATGGCCAGCCACCCCGTGAGCCTGCACGCCGTGGGCGTGAGCTACTGGAAGGCCAGCGAGGGCGCCGAGTACGACGACCAGACCAGCCAGCGCGAGAAGGAGGACGACAAGGTGTTCCCCGGCGGCAGCCACACCTACGTGTGGCAGGTGCTGAAGGAGAACGGCCCCATGGCCAGCGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCACGTGGACCTGGTGAAGGACCTGAACAGCGGCCTGATCGGCGCCCTGCTGGTGTGCCGCGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACCAAGAACAGCCTGATGCAGGACCGCGACGCCGCCAGCGCCCGCGCCTGGCCCAAGATGCACACCGTGAACGGCTACGTGAACCGCAGCCTGCCCGGCCTGATCGGCTGCCACCGCAAGAGCGTGTACTGGCACGTGATCGGCATGGGCACCACCCCCGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTGCGCAACCACCGCCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACCGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCACGACGGCATGGAGGCCTACGTGAAGGTGGACAGCTGCCCCGAGGAGCCCCAGCTGCGCATGAAGAACAACGAGGAGGCCGAGGACTACGACGACGACCTGACCGACAGCGAGATGGACGTGGTGCGCTTCGACGACGACAACAGCCCCAGCTTCATCCAGATCCGCAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATCGCCGCCGAGGAGGAGGACTGGGACTACGCCCCCCTGGTGCTGGCCCCCGACGACCGCAGCTACAAGAGCCAGTACCTGAACAACGGCCCCCAGCGCATCGGCCGCAAGTACAAGAAGGTGCGCTTCATGGCCTACACCGACGAGACCTTCAAGACCCGCGAGGCCATCCAGCACGAGAGCGGCATCCTGGGCCCCCTGCTGTACGGCGAGGTGGGCGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCCGCCCCTACAACATCTACCCCCACGGCATCACCGACGTGCGCCCCCTGTACAGCCGCCGCCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCCATCCTGCCCGGCGAGATCTTCAAGTACAAGTGGACCGTGACCGTGGAGGACGGCCCCACCAAGAGCGACCCCCGCTGCCTGACCCGCTACTACAGCAGCTTCGTGAACATGGAGCGCGACCTGGCCAGCGGCCTGATCGGCCCCCTGCTGATCTGCTACAAGGAGAGCGTGGACCAGCGCGGCAACCAGATCATGAGCGACAAGCGCAACGTGATCCTGTTCAGCGTGTTCGACGAGAACCGCAGCTGGTACCTGACCGAGAACATCCAGCGCTTCCTGCCCAACCCCGCCGGCGTGCAGCTGGAGGACCCCGAGTTCCAGGCCAGCAACATCATGCACAGCATCAACGGCTACGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACATCCTGAGCATCGGCGCCCAGACCGACTTCCTGAGCGTGTTCTTCAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGACACCCTGACCCTGTTCCCCTTCAGCGGCGAGACCGTGTTCATGAGCATGGAGAACCCCGGCCTGTGGATCCTGGGCTGCCACAACAGCGACTTCCGCAACCGCGGCATGACCGCCCTGCTGAAGGTGAGCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTACGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAACGCCATCGAGCCCCGCAGCTTCAGCCAGAACCCCCCCGTGCTGAAGCGCCACCAGCGCGAGATCACCCGCACCACCCTGCAGAGCGACCAGGAGGAGATCGACTACGACGACACCATCAGCGTGGAGATGAAGAAGGAGGACTTCGACATCTACGACGAGGACGAGAACCAGAGCCCCCGCAGCTTCCAGAAGAAGACCCGCCACTACTTCATCGCCGCCGTGGAGCGCCTGTGGGACTACGGCATGAGCAGCAGCCCCCACGTGCTGCGCAACCGCGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCCCTGTACCGCGGCGAGCTGAACGAGCACCTGGGCCTGCTGGGCCCCTACATCCGCGCCGAGGTGGAGGACAACATCATGGTGACCTTCCGCAACCAGGCCAGCCGCCCCTACAGCTTCTACAGCAGCCTGATCAGCTACGAGGAGGACCAGCGCCAGGGCGCCGAGCCCCGCAAGAACTTCGTGAAGCCCAACGAGACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCAGCGACGTGGACCTGGAGAAGGACGTGCACAGCGGCCTGATCGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCCGCCCACGGCCGCCAGGTGACCGTGCAGGAGTTCGCCCTGTTCTTCACCATCTTCGACGAGACCAAGAGCTGGTACTTCACCGAGAACATGGAGCGCAACTGCCGCGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACCGCTTCCACGCCATCAACGGCTACATCATGGACACCCTGCCCGGCCTGGTGATGGCCCAGGACCAGCGCATCCGCTGGTACCTGCTGAGCATGGGCAGCAACGAGAACATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGCAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCCGGCGTGTTCGAGACCGTGGAGATGCTGCCCAGCAAGGCCGGCATCTGGCGCGTGGAGTGCCTGATCGGCGAGCACCTGCACGCCGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCAGCGGCCACATCCGCGACTTCCAGATCACCGCCAGCGGCCAGTACGGCCAGTGGGCCCCCAAGCTGGCCCGCCTGCACTACAGCGGCAGCATCAACGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCACGGCATCAAGACCCAGGGCGCCCGCCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACCGCGGCAACAGCACCGGCACCCTGATGGTGTTCTTCGGCAACGTGGACAGCAGCGGCATCAAGCACAACATCTTCAACCCCCCCATCATCGCCCGCTACATCCGCCTGCACCCCACCCACTACAGCATCCGCAGCACCCTGCGCATGGAGCTGATGGGCTGCGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCAGCGACGCCCAGATCACCGCCAGCAGCTACTTCACCAACATGTTCGCCACCTGGAGCCCCAGCAAGGCCCGCCTGCACCTGCAAGGCCGCAGCAACGCCTGGCGCCCCCAGGTGAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACCGGCGTGACCACCCAGGGCGTGAAGAGCCTGCTGACCAGCATGTACGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCCGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCCGCTACCTGCGCATCCACCCCCAGAGCTGGGTGCACCAGATCGCCCTGCGCATGGAGGTGCTGGGCTGCGAGGCCCAGGACCTGTACTAAHuman Full 7056 323 391ATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGLength FVIIICGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTORF (codonGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATAoptimized)TCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCTGGAATCCGGCCTGATGAACAGCCAAGAGTCCTCCTGGGGCAAGAATGTGTCCAGCACCGAGTCCGGCAGACTGTTCAAGGGAAAGAGAGCCCACGGACCTGCTCTGCTGACCAAGGATAACGCCCTGTTCAAAGTGTCCATCAGCCTGCTCAAGACCAACAAGACCTCCAACAACTCCGCCACCAACAGAAAGACCCACATCGACGGCCCTAGCCTGCTGATCGAGAATAGCCCTAGCGTCTGGCAGAATATCCTGGAAAGCGACACCGAGTTCAAGAAAGTGACCCCTCTGATCCACGACCGGATGCTCATGGACAAGAACGCCACCGCTCTGCGGCTGAACCACATGAGCAACAAGACAACCAGCAGCAAGAATATGGAAATGGTGCAGCAGAAGAAAGAGGGCCCCATTCCTCCAGACGCTCAGAACCCCGATATGAGCTTCTTCAAGATGCTCTTTCTGCCCGAGAGCGCCCGGTGGATTCAGAGAACACACGGCAAGAACTCCCTGAACTCCGGCCAGGGACCTTCTCCAAAGCAGCTGGTTTCCCTGGGACCTGAGAAGTCCGTGGAAGGACAGAACTTCCTGAGCGAAAAGAACAAAGTGGTCGTCGGCAAGGGCGAGTTCACCAAGGATGTGGGCCTGAAAGAGATGGTCTTTCCCAGCAGCCGGAACCTGTTCCTGACCAACCTGGACAACCTGCACGAGAACAACACCCACAATCAAGAGAAGAAGATCCAAGAGGAAATCGAAAAGAAAGAGACACTCATCCAAGAGAACGTGGTGCTGCCTCAGATCCACACAGTGACCGGCACCAAGAACTTTATGAAGAATCTGTTCCTGCTGAGTACCCGGCAGAACGTGGAAGGCAGCTACGATGGCGCTTATGCCCCTGTGCTGCAGGACTTCAGATCCCTGAACGACTCCACCAATCGGACAAAGAAGCACACAGCCCACTTCTCCAAGAAGGGCGAAGAAGAGAACCTGGAAGGACTGGGCAATCAGACCAAGCAGATCGTCGAGAAGTACGCCTGCACCACCAGAATCAGCCCCAACACAAGCCAGCAGAACTTCGTGACCCAGCGGAGCAAAAGAGCCCTGAAGCAGTTCGGCTGCCCCTGGAAGAAACCGAGCTGGAAAAGCGGATCATCGTGGACGACACCAGCACACAGTGGTCCAAGAACATGAAGCACTTGACCCCTAGCACACTGACCCAGATCGACTACAACGAGAAAGAGAAGGGCGCTATCACACAGAGCCCACTGAGCGACTGTCTGACCAGAAGCCACAGCATCCCTCAGGCCAACAGATCCCCTCTGCCAATCGCCAAAGTGTCTAGCTTCCCCAGCATCAGACCCATCTACCTGACCAGAGTGCTGTTCCAGGACAACAGCAGCCATCTGCCAGCCGCCAGCTACCGGAAGAAAGATTCTGGCGTGCAAGAGAGCAGCCACTTTCTGCAGGGCGCTAAGAAGAACAATCTGAGCCTGGCTATTCTGACCCTGGAAATGACCGGCGATCAGAGAGAAGTCGGCTCTCTGGGCACCAGCGCCACAAATAGCGTGACCTACAAAAAGGTGGAAAACACCGTGCTGCCTAAGCCTGACCTGCCAAAGACAAGCGGCAAGGTGGAACTGCTGCCAAAGGTGCACATCTACCAGAAGGACCTGTTTCCTACCGAGACAAGCAACGGCTCTCCCGGCCATCTGGATCTGGTGGAAGGATCTCTGCTGCAGGGAACCGAGGGCGCCATCAAGTGGAACGAGGCCAATAGACCTGGCAAGGTGCCCTTCCTGAGAGTGGCCACAGAGTCCAGCGCCAAGACACCCTCTAAACTGCTGGACCCTCTGGCCTGGGACAACCACTATGGCACTCAGATCCCCAAAGAGGAATGGAAGTCCCAAGAGAAGTCCCCTGAAAAGACCGCCTTCAAGAAGAAGGACACCATTCTGTCCCTGAATGCCTGCGAGAGCAACCACGCCATTGCCGCCATCAATGAGGGCCAGAACAAGCCCGAGATCGAAGTGACCTGGGCCAAGCAGGGAAGAACCGAGAGACTGTGTAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGAGAGAAATCACCCGGACCACACTGCAGAGCGACCAAGAAGAGATCGATTACGACGATACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCTCGGAGCTTCCAGAAGAAAACCAGGCACTACTTTATTGCCGCCGTCGAGCGGCTGTGGGACTACGGAATGTCTAGCTCTCCTCACGTGCTGCGGAATAGAGCCCAGTCTGGTAGCGTGCCCCAGTTCAAAAAGGTCGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCAGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCATATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGACCTCGAAAAGGACGTGCACAGCGGACTCATCGGCCCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTTTTCACCATCTTCGACGAAACGAAGTCCTGGTACTTCACCGAAAACATGGAAAGAAACTGCAGGGCCCCTTGCAACATTCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACTCCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTTGAGATGCTGCCTAGCAAGGCCGGAATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGGATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGCCACATTAGAGACTTCCAGATCACCGCCAGCGGACAGTATGGACAGTGGGCCCCTAAACTGGCCAGACTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCTTTCAGCTGGATCAAAGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAAAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTGATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCAGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCTCTAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCTAAGGCCCGGCTTCATCTGCAAGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAATGGCTCCAGGTGGACTTTCAGAAAACCATGAAAGTGACAGGCGTGACCACACAGGGCGTCAAGTCCCTGCTGACCTCTATGTACGTGAAAGAGTTTCTGATCAGCTCCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGAAATCAGGACAGCTTCACACCCGTGGTCAATAGTCTGGACCCACCACTGCTGACCCGCTACCTGCGAATTCACCCTCAGTCTTGGGTGCACCAGATTGCCCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGCodon Optimized 4980 241 392ATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGHuman FactorCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTVIII 206-GCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATAvariant ORFTCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCCTCCTGTGCTGAAGCGGCACCAGCGGGAAATCACCAGAACCACACTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGATCCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGGGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCAAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATTAGCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAG CpG-free human 4374   1 393ATGCAGATAGAATTATCTACTTGTTTTTTTCTTTGTCTGCTCAGGTTCTGCTTCTCTGCTACAAGGAGGTATTACTTAGGThFVIII BDDGCTGTAGAGTTATCTTGGGACTACATGCAGTCTGATCTTGGGGAGCTCCCTGTGGATGCAAGATTTCCCCCTAGAGTACCCOOL optimizedTAAAAGTTTCCCTTTCAACACAAGTGTGGTGTATAAGAAGACTCTGTTTGTTGAGTTCACAGATCACCTTTTCAATATTGCCAAGCCCAGACCCCCCTGGATGGGTCTACTGGGGCCAACAATTCAAGCAGAGGTATATGATACAGTGGTGATCACATTAAAGAACATGGCATCCCACCCTGTAAGCCTGCATGCTGTTGGTGTGTCCTATTGGAAAGCAAGTGAAGGTGCTGAATATGATGATCAAACATCCCAAAGAGAGAAGGAAGATGACAAAGTCTTCCCAGGTGGCTCTCACACCTATGTGTGGCAAGTGCTCAAGGAAAATGGGCCTATGGCTAGTGATCCATTGTGCCTCACATATAGCTACCTGTCCCATGTGGACTTAGTTAAAGATCTGAACTCAGGTCTGATTGGTGCCCTGCTGGTCTGTAGGGAAGGATCACTAGCTAAAGAAAAAACCCAGACTTTACACAAGTTCATCTTATTATTTGCTGTTTTTGATGAGGGAAAGAGCTGGCACTCAGAGACTAAGAATTCACTCATGCAGGATAGAGATGCAGCTTCTGCTAGAGCATGGCCAAAAATGCATACTGTGAATGGGTATGTTAACAGGAGTCTGCCTGGCCTCATAGGCTGCCATAGAAAATCAGTGTACTGGCATGTCATAGGTATGGGCACTACTCCTGAGGTGCATTCCATTTTTTTGGAGGGGCATACTTTCCTTGTAAGAAACCACAGGCAGGCTTCATTGGAAATTAGTCCAATAACCTTTCTCACAGCCCAGACTCTGCTAATGGACTTGGGGCAGTTTTTATTATTTTGTCACATTAGCTCTCATCAGCATGATGGAATGGAAGCCTATGTTAAGGTTGATTCCTGCCCAGAAGAGCCCCAGTTGAGAATGAAGAACAATGAGGAAGCAGAAGACTATGATGATGATCTAACAGACTCAGAAATGGATGTTGTGAGATTTGATGATGATAACTCCCCAAGTTTTATTCAGATCAGGTCAGTGGCCAAGAAACATCCAAAAACATGGGTTCATTATATTGCAGCAGAAGAAGAAGACTGGGATTATGCACCCTTGGTATTAGCCCCTGATGACAGAAGCTATAAGTCACAATATCTCAACAATGGACCCCAGAGAATTGGCAGAAAGTACAAAAAAGTCAGGTTTATGGCTTATACTGATGAGACTTTCAAAACTAGAGAGGCTATTCAACATGAATCTGGCATCCTAGGTCCTTTGTTATATGGGGAAGTAGGGGACACCTTATTAATTATATTTAAAAACCAGGCCAGTAGGCCTTACAACATTTACCCCCACGGTATAACAGATGTCAGACCACTGTACTCAAGGAGGCTACCAAAAGGGGTGAAGCACCTAAAAGACTTCCCCATCCTACCTGGTGAGATTTTCAAGTATAAGTGGACAGTGACTGTAGAAGATGGTCCAACCAAATCAGATCCCAGATGCCTGACAAGGTACTACAGTAGCTTTGTAAATATGGAGAGAGACCTGGCCTCAGGGTTAATAGGGCCCCTACTGATCTGCTACAAGGAGAGTGTAGACCAAAGAGGCAACCAAATTATGTCAGACAAGAGAAATGTCATCCTCTTTAGTGTTTTTGATGAGAATAGGTCCTGGTACCTGACAGAAAATATCCAAAGATTCCTTCCAAATCCAGCAGGGGTACAACTGGAGGACCCTGAGTTCCAGGCCTCCAATATCATGCACAGCATCAATGGTTATGTGTTTGACAGTTTGCAACTTTCAGTGTGCCTTCATGAGGTTGCATACTGGTATATACTTTCTATAGGGGCCCAGACAGACTTCTTGTCAGTTTTCTTTTCAGGCTATACTTTCAAGCATAAGATGGTATATGAAGATACCCTGACTCTTTTTCCCTTTAGTGGTGAGACAGTTTTCATGTCAATGGAAAACCCAGGGCTTTGGATCTTGGGATGCCACAACTCTGACTTTAGAAACAGAGGCATGACTGCCCTTTTAAAGGTGTCCTCTTGTGATAAGAATACTGGAGACTACTATGAGGACAGCTATGAGGACATTTCAGCCTATCTGCTGTCAAAAAATAATGCTATAGAGCCAAGGTCATTTTCTCAAAATCCTCCTGTCCTGAAGAGGCACCAAAGAGAGATAACCAGGACTACCTTACAGTCTGACCAGGAGGAAATTGATTATGATGACACAATAAGTGTGGAGATGAAAAAGGAGGATTTTGATATTTATGATGAGGATGAAAACCAATCTCCAAGATCTTTCCAGAAGAAGACTAGACACTATTTCATTGCTGCAGTGGAAAGACTGTGGGACTATGGAATGTCCTCCAGCCCTCATGTTCTCAGGAATAGGGCTCAAAGTGGTTCTGTGCCACAGTTCAAGAAGGTGGTGTTTCAGGAATTCACAGATGGGTCCTTTACTCAACCACTGTATAGGGGAGAACTCAATGAACATCTAGGATTACTAGGTCCCTACATTAGAGCTGAGGTGGAGGACAATATCATGGTCACTTTCAGGAACCAGGCATCTAGACCATACTCTTTTTACAGCTCCTTGATATCTTATGAAGAGGACCAGAGACAGGGTGCAGAACCCAGGAAGAACTTTGTGAAACCCAATGAGACCAAAACATACTTCTGGAAGGTCCAGCATCATATGGCTCCTACCAAGGATGAATTTGACTGCAAAGCCTGGGCTTACTTCTCAGATGTGGATTTGGAGAAAGATGTGCACAGTGGCTTGATTGGGCCTCTTTTGGTATGTCATACCAACACCTTAAACCCTGCTCATGGAAGACAAGTAACAGTGCAAGAGTTTGCCCTCTTTTTCACTATATTTGATGAGACAAAAAGTTGGTACTTTACTGAGAATATGGAGAGGAACTGCAGGGCTCCCTGCAATATACAGATGGAGGATCCTACTTTCAAGGAAAACTATAGGTTTCATGCCATTAATGGCTATATCATGGATACATTGCCTGGCTTAGTTATGGCCCAGGATCAGAGAATCAGATGGTACCTCCTTAGTATGGGAAGCAATGAAAACATACATTCTATTCACTTCAGTGGACATGTCTTTACTGTGAGAAAAAAGGAAGAGTACAAGATGGCACTGTACAATCTGTACCCTGGAGTGTTTGAGACAGTTGAGATGCTGCCCAGCAAGGCTGGGATCTGGAGGGTAGAATGCCTGATGGAGAACACTTACATGCAGGTATGTCCACTCTATTCTTGGTCTATTCCAACAAGTGTCAGACCCCCCTAGGCATGGCATCTGGCCATATTAGAGATTTCCAGATAACTGCTTCTGGTCAGTATGGCCAGTGGGCCCCCAAGCTGCTAGACTTCACTACAGTGGTTCAATAAATGCCTGGTCCACCAAGGAGCCCTTCAGCTGGATTAAAGTGGACTTGCTAGCCCCCATGATCATTCATGGGATAAAAACACAGGGTGCCAGACAGAAATTTTCATCTCTGTACATCAGCCAGTTTATCATCATGTACAGTCTGGATGGCAAAAAATGGCAGACCTACAGAGGTAACTCTACAGGTACACTAATGGTATTTTTTGGCAATGTAGATTCATCAGGTATCAAGCATAATATCTTTAATCCTCCCATTATTGCCAGATACATCAGGTTGCACCCAACTCATTACAGTATAAGATCCACTTTAAGGATGGAGCTTATGGGGTGTGACCTTAATAGCTGCAGTATGCCCTTGGGAATGGAAAGTAAGGCCATATCAGATGCCCAGATTACTGCAAGCAGCTACTTTACAAACATGTTTGCAACCTGGTCTCCTAGTAAAGCCAGGCTGCACTTGCAGGGTAGGAGCAATGCATGGAGACCCCAGGTCAATAACCCAAAAGAGTGGCTTCAAGTTGACTTTCAAAAGACAATGAAGGTAACAGGGGTAACCACCCAAGGGGTAAAGAGTCTGCTGACATCTATGTATGTAAAAGAATTCCTCATCTCAAGTAGTCAAGATGGGCACCAGTGGACACTGTTCTTCCAGAATGGAAAAGTTAAGGTGTTCCAAGGTAATCAAGACAGCTTCACTCCAGTGGTGAACAGTCTGGATCCACCACTCCTAACCAGGTACCTGAGGATCCACCCTCAGTCCTGGGTCCACCAGATAGCTCTGAGAATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTATACTGA CpG-free human 7056   0 394ATGCAAATTGAATTGAGTACCTGTTTTTTCCTCTGTCTATTAAGGTTTTGTTTCAGTGCAACAAGAAGGTATTATCTGGGAhFVIII FullGCTGTTGAGTTATCCTGGGACTACATGCAGTCTGACCTGGGAGAGCTCCCAGTGGATGCTAGGTTTCCCCCAAGAGTACLength COOLCCAAAAGCTTCCCTTTCAACACTTCTGTTGTCTATAAAAAGACTCTGTTTGTAGAATTCACAGATCATCTGTTTAACATTGCoptimizedCAAGCCTAGACCTCCATGGATGGGGCTGCTGGGACCAACTATTCAGGCAGAGGTTTATGACACTGTGGTGATTACACTTAAGAATATGGCATCCCACCCAGTGAGCCTGCATGCTGTTGGGGTTTCCTACTGGAAGGCAAGTGAAGGAGCTGAGTATGATGACCAGACTAGCCAAAGAGAGAAGGAGGATGACAAGGTCTTTCCAGGTGGCAGCCACACCTATGTCTGGCAGGTGCTGAAGGAGAATGGCCCTATGGCCTCTGATCCTTTGTGTCTAACCTACAGTTATTTGTCTCATGTGGACCTGGTGAAGGACCTTAACTCAGGGCTGATAGGGGCCTTGCTGGTGTGTAGGGAGGGAAGCCTGGCTAAGGAAAAGACACAGACTCTGCATAAGTTTATTCTCTTGTTTGCAGTGTTTGATGAGGGTAAAAGCTGGCACTCTGAAACAAAAAACAGCTGATGCAGGACAGGGATGCTGCCAGTGCTAGGGCTGGCCTAAGATGCACACAGTCAATGGCTATGTTAACAGATCACTGCCTGGTTTGATTGGCTGCCACAGAAAGTCTGTTTACTGGCATGTAATTGGGATGGGGACAACCCCAGAAGTTCACTCTATCTTCCTGGAAGGGCACACCTTCCTGGTTAGAAATCATAGACAGGCATCCTTAGAGATTTCTCCTATTACATTCCTGACTGCCCAGACCCTCCTCATGGACCTGGGTCAGTTCCTGCTTTTTTGCCACATTTCCTCCCACCAGCATGATGGCATGGAGGCCTATGTGAAAGTTGATTCTTGCCCTGAGGAGCCTCAGCTTAGGATGAAGAACAATGAAGAGGCTGAGGACTATGATGATGACCTAACAGACAGTGAGATGGATGTTGTGAGGTTTGATGATGACAATTCACCTAGTTTCATCCAGATTAGGTCTGTGGCTAAGAAACATCCAAAGACCTGGGTTCACTACATTGCAGCAGAGGAGGAGGACTGGGATTATGCCCCCCTGGTTCTGGCACCTGATGACAGAAGCTATAAGTCCCAGTATCTCAACAATGGCCCCCAGAGGATTGGAAGAAAATACAAAAAAGTCAGATTTATGGCCTATACAGATGAGACTTTTAAAACTAGGGAAGCTATTCAGCATGAGTCAGGGATCCTGGGACCTCTTCTTTATGGAGAGGTGGGGGACACTCTTCTGATAATTTTTAAAAATCAGGCAAGTAGACCTTATAACATCTACCCCCATGGGATCACTGATGTCAGGCCTCTGTACTCAAGAAGGCTGCCCAAGGGTGTCAAACACCTCAAGGATTTCCCTATTCTTCCTGGGGAGATCTTCAAATACAAGTGGACAGTTACTGTGGAGGATGGCCCCACCAAATCTGACCCAAGGTGCCTCACCAGATATTACAGCTCCTTTGTGAATATGGAAAGAGACCTAGCTAGTGGTCTCATTGGCCCACTGCTCATCTGTTACAAGGAGTCAGTGGACCAGAGAGGGAATCAAATTATGTCTGACAAGAGAAATGTGATCCTTTTCTCAGTGTTTGATGAGAACAGAAGCTGGTATCTGACAGAGAACATTCAAAGGTTTCTGCCAAACCCAGCTGGAGTTCAGCTGGAGGACCCTGAATTCCAGGCATCTAATATTATGCATTCTATCAATGGATATGTGTTTGATAGTCTGCAGCTGTCTGTGTGCCTGCATGAAGTGGCCTACTGGTATATCCTCTCTATTGGTGCCCAGACAGATTTCTTGTCTGTGTTTTTCAGTGGTTACACATTTAAACATAAAATGGTGTATGAGGACACACTTACATTATTCCCTTTTAGTGGAGAGACAGTGTTTATGTCCATGGAGAATCCAGGCCTGTGGATCTTGGGGTGTCATAATAGTGACTTTAGGAATAGAGGTATGACAGCTCTGCTCAAAGTGTCTTCCTGTGACAAGAACACAGGGGACTATTATGAGGACTCTTATGAGGACATTTCAGCCTATTTACTGTCCAAGAACAATGCCATTGAACCAAGATCTTTCTCTCAGAACTCAAGACACCCCAGCACCAGGCAGAAGCAGTTCAATGCAACCACCATCCCAGAGAATGATATTGAAAAAACTGACCCTTGGTTTGCCCACAGGACTCCCATGCCTAAGATTCAGAATGTATCTTCATCAGACTTGCTGATGCTCCTGAGACAGAGCCCAACTCCTCATGGCCTGAGCTTAAGTGACCTCCAGGAGGCTAAGTATGAAACTTTCTCAGATGATCCCAGTCCTGGTGCCATTGATTCTAATAATTCCCTGTCTGAGATGACACACTTCAGACCCCAGCTCCACCACTCTGGAGATATGGTCTTTACTCCTGAATCAGGCCTGCAGCTGAGGCTGAATGAAAAGCTGGCACCACAGCAGCTACTGAACTGAAAAAGCTGGATTTTAAAGTGAGCTCCACATCCAACAACCTGATTTCCACTATCCCCTCAGATAACTTGGCAGCAGGGACTGATAACACAAGCTCACTGGGTCCCCCTTCCATGCCTGTCCATTATGATAGTCAGCTGGATACTACTCTGTTTGGTAAGAAATCCTCTCCCCTGACTGAAAGTGGAGGCCCTCTTTCCCTGTCAGAGGAGAACAATGACAGCAAACTGTTAGAGTCTGGACTCATGAACTCACAGGAAAGTTCTTGGGGCAAGAATGTGTCTAGCACTGAGTCTGGGAGACTGTTTAAGGGCAAGAGGGCCCATGGACCAGCCCTGCTGACAAAGGATAATGCACTGTTTAAGGTCTCAATCAGCCTGTTGAAGACCAACAAAACCAGTAATAATTCTGCAACAAACAGGAAGACCCACATTGATGGACCCTCTCTTCTCATTGAGAATTCCCCTTCTGTCTGGCAGAACATCTTGGAATCAGATACAGAGTTCAAGAAGGTGACTCCCCTGATCCATGATAGGATGCTCATGGATAAGAATGCCACAGCACTCAGGCTCAATCACATGAGCAACAAGACTACCTCCTCCAAAAATATGGAGATGGTGCAGCAGAAGAAGGAAGGTCCCATTCCCCCTGATGCACAGAATCCAGATATGAGCTTCTTTAAGATGCTGTTTTTCCTGAGTCTGCCAGGTGGATTCAGAGGACACATGGGAAGAACTCACTTAACTCTGGCCAAGGTCCCAGTCCTAAGCAGTTGGTGAGCTGGGGCCTGAGAAAAGTGTGGAGGGACAGAATTTCCTGTCTGAAAAGAACAAAGTGGTTGTGGGCAAAGGAGAGTTCACTAAAGATGTGGGACTGAAGGAGATGGTGTTTCCTAGTTCAAGAAATCTCTTTCTTACCAATCTTGATAACCTGCATGAGAACAACACCCACAATCAGGAAAAGAAGATCCAGGAGGAAATTGAGAAGAAGGAAACCCTGATCCAGGAAAATGTTGTGCTGCCACAGATCCACACTGTTACTGGGACCAAAAATTTCATGAAAAACCTGTTTCTCCTGTCTACCAGACAGAATGTAGAGGGAAGCTATGATGGGGCCTATGCCCCTGTTCTCCAGGACTTCAGATCCCTGAATGACTCCACCAACAGGACTAAGAAACATACTGCACACTTTAGTAAGAAGGGGGAGGAGGAGAACCTGGAGGGATTGGGAAACCAGACCAAGCAGATTGTGGAGAAATATGCTGCACCACTAGAATCTCACCTAACACAAGCCAGCAGAACTTTGTGACCCAGAGGTCTAAGAGGGCCCTCAAACAGTTTAGATTGCCTTTGGAGGAGACTGAGCTGGAAAAGAGGATTATTGTGGATGACACCTCTACCCAGTGGAGCAAGAATATGAAGCACCTGACCCCAAGCACCCTTACTCAGATTGATTACAATGAAAAAGAAAAGGGTGCCATCACCCAGAGCCCCTTGAGTGACTGTTTGACAAGGTCCCACAGTATCCCACAGGCTAACAGATCTCCTCTGCCTATTGCCAAGGTCAGCTCCTTCCCTTCCATCAGACCCATCTATCTGACCAGAGTGCTGTTCCAGGACAACTCCTCTCATCTGCCAGCTGCCTCCTATAGGAAGAAGGATAGTGGTGTGCAGGAATCATCTCACTTTCTGCAGGGAGCCAAGAAGAACAATCTGAGCCTGGCAATTCTCACACTTGAGATGACAGGAGACCAGAGGGAAGTGGGAAGTCTGGGCACTTCTGCTACCAATTCTGTCACCTACAAGAAAGTGGAGAATACAGTTCTGCCCAAGCCAGACCTGCCTAAAACATCTGGAAAGGTTGAGCTGCTGCCTAAGGTGCACATATACCAGAAGGACCTATTCCCTACAGAGACATCCAATGGCTCCCCAGGCCACCTGGACTTGGTAGAGGGGTCTCTCCTGCAGGGCACTGAAGGTGCTATCAAGTGGAATGAGGCTAATAGACCTGGGAAGGTGCCATTTCTGAGGGTGGCCACAGAGAGCTCAGCTAAGACACCCAGCAAGCTGCTTGATCCCCTGGCCTGGGACAATCACTATGGAACCCAGATTCCTAAGGAGGAGTGGAAGTCCCAGGAGAAGTCCCCAGAGAAGACTGCTTTCAAGAAGAAGGACACTATCCTCTCCCTGAATGCCTGTGAGTCTAACCATGCTATTGCTGCCATCAATGAAGGCCAGAACAAGCCTGAAATTGAGGTGACATGGGCCAAGCAGGGCAGAACAGAGAGGCTGTGCAGCCAGAACCCACCTGTGCTCAAGAGGCACCAGAGAGAGATCACTAGAACCACTCTGCAGTCAGACCAGGAAGAGATTGACTATGATGACACCATCTCAGTTGAGATGAAAAAGGAGGATTTTGATATTTATGATGAGGATGAGAATCAATCCCCAAGATCTTTTCAGAAGAAGACAAGACATTACTTCATTGCTGCAGTGGAGAGACTGTGGGATTATGGCATGTCCTCCTCACCTCATGTGCTGAGAAATAGAGCCCAGTCTGGCTCTGTGCCCCAGTTCAAAAAAGTGGTGTTTCAGGAGTTCACAGATGGTTCCTTCACACAGCCCCTGTACAGGGGTGAGCTGAATGAGCACCTGGGGTTGCTGGGCCCCTACATCAGAGCTGAAGTTGAGGATAATATTATGGTGACCTTCAGGAATCAGGCCAGTAGGCCTTACTCTTTCTACTCCAGCCTGATCTCATATGAGGAGGACCAGAGACAGGGGGCTGAACCTAGGAAGAATTTTGTGAAGCCCAATGAAACAAAGACATACTTCTGGAAGGTGCAGCACCACATGGCTCCTACCAAGGATGAATTTGATTGCAAGGCTTGGGCCTATTTTTCTGATGTGGACCTGGAGAAGGATGTGCATTCTGGCCTCATTGGCCCTCTCTTGGTGTGTCACACCAATACCTTGAATCCAGCCCATGGCAGACAGGTGACTGTTCAGGAGTTTGCTCTGTTTTTCACCATCTTTGATGAGACAAAGTCCTGGTATTTTACAGAGAATATGGAGAGGAATTGCAGGGCCCCCTGCAACATCCAGATGGAAGACCCAACCTTCAAGGAGAACTACAGATTCCATGCAATCAATGGATATATCATGGACACCCTGCCTGGCCTGGTTATGGCTCAGGATCAGAGAATCAGGTGGTATCTGCTGTCTATGGGGTCAAATGAGAACATCCATAGTATCCACTTCTCTGGCCATGTGTTTACAGTGAGAAAGAAGGAAGAATATAAGATGGCCCTGTACAACCTCTACCCTGGAGTGTTTGAAACTGTTGAAATGCTGCCTTCAAAGGCTGGAATCTGGAGGGTGGAATGCCTGATTGGAGAGCACCTTCATGCTGGCATGTCCACCCTGTTTCTGGTGTACAGCAATAAGTGTCAGACTCCTCTGGGTATGGCTTCAGGCCACATCAGGGACTTCCAGATCACTGCCTCAGGACAGTATGGACAGTGGGCTCCCAAACTGGCAAGGCTCCACTACTCAGGCAGCATTAATGCCTGGTCCACCAAAGAGCCTTTCAGCTGGATTAAGGTGGATCTCCTGGCACCTATGATCATCCATGGGATCAAGACTCAGGGAGCTAGGCAGAAGTTTAGCAGCCTGTACATTTCTCAGTTTATCATTATGTACAGCCTGGATGGAAAAAAGTGGCAGACCTATAGAGGAAACTCCACTGGAACACTGATGGTGTTTTTTGGCAATGTGGACTCAAGTGGGATCAAGCACAATATCTTCAACCCCCCTATCATTGCCAGATACATCAGGCTCCACCCTACACACTACTCTATCAGATCTACACTTAGGATGGAGCTGATGGGATGTGATCTGAACTCTTGCAGTATGCCTCTGGGTATGGAGTCCAAGGCCATCTCTGATGCTCAGATCACAGCCAGCTCTTACTTTACTAATATGTTTGCTACCTGGTCCCCAAGCAAGGCAAGACTGCACCTGCAGGGCAGATCCAATGCTTGGAGACCCCAGGTGAATAACCCAAAAGAGTGGCTTCAGGTGGACTTCCAGAAGACTATGAAGGTGACTGGAGTGACCACCCAGGGGGTGAAGTCACTGCTGACCTCCATGTATGTGAAGGAGTTCCTGATCTCCAGCTCCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGAAAGGTGAAGGTGTTCCAGGGAAACCAGGACTCCTTTACCCCTGTGGTGAACTCCCTGGACCCCCCACTGCTGACCAGATACCTGAGAATCCATCCCCAGAGCTGGGTGCACCAGATTGCCCTTAGGATGGAGGTGCTGGGATGTGAAGCCCAGGACCTGTACTAA CpG-free human 5013 https://   0 395ATGCAAATTGAGTTAAGTACCTGTTTTTTCCTGTGCCTGTTGAGATTTTGCTTCTCTGCTACCAGAAGGTATTACCTGGGAhFVIII 226 www.ncbi.GCTGTGGAGCTGTCATGGGACTACATGCAGTCTGACCTGGGAGAGCTCCCTGTTGATGCTAGGTTTCCCCCTAGGGTGCVariant COOL nLm.nih.CAAAGTCCTTTCCTTTTAACACCTCAGTGGTGTACAAGAAGACACTGTTTGTGGAGTTTACTGACCACCTCTTCAATATTGoptimized gov/CTAAGCCAAGACCCCCTTGGATGGGTCTCTTGGGCCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTGATTACCCTCpubmed/AAGAATATGGCTTCTCATCCTGTTAGTCTGCATGCAGTAGGGGTCAGCTATTGGAAGGCCAGTGAAGGAGCTGAGTAT14726380GATGACCAGACCAGCCAGAGAGAGAAAGAGGATGACAAGGTGTTCCCTGGGGGATCACACACCTATGTGTGGCAGGTGTTGAAGGAAAATGGACCCATGGCCTCTGACCCACTCTGCCTGACTTACTCCTACCTGTCTCATGTGGACCTGGTCAAAGATCTGAACTCTGGCCTGATTGGGGCACTGTTGGTGTGCAGAGAAGGGTCTCTTGCCAAGGAGAAGACTCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAAGGCAAGTCTTGGCATTCTGAGACTAAGAACAGCCTGATGCAAGATAGAGATGCTGCATCTGCCAGGGCCTGGCCTAAGATGCACACTGTGAATGGATATGTGAATAGGTCCTTGCCTGGCCTGATTGGATGCCATAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTTCACTCTATTTTTCTGGAGGGCCATACTTTCCTTGTGAGGAATCATAGACAGGCCTCTCTGGAGATTAGCCCTATTACATTTCTGACTGCTCAGACCCTCCTCATGGACTTGGGGCAGTTTCTGTTATTCTGCCACATCAGCAGCCATCAACATGATGGTATGGAAGCCTATGTCAAAGTGGATAGCTGCCCTGAAGAACCACAGCTTAGGATGAAGAACAATGAGGAGGCAGAGGACTATGATGATGATCTGACTGATTCTGAAATGGATGTGGTGAGATTTGATGATGATAATAGCCCTTCCTTTATCCAGATCAGGTCAGTGGCCAAGAAGCACCCAAAGACCTGGGTGCATTATATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCTCTGGTGCTGGCCCCTGATGATAGAAGCTATAAGTCTCAATACTTGAATAATGGCCCACAGAGGATTGGGAGAAAGTACAAAAAGGTGAGATTTATGGCCTACACTGATGAGACTTTCAAGACCAGAGAGGCCATCCAGCATGAATCAGGCATTTTGGGTCCCCTGCTGTATGGAGAGGTGGGGGATACCCTGCTGATCATCTTTAAGAACCAGGCCAGCAGACCTTACAACATTTACCCTCATGGGATCACAGATGTGAGGCCACTCTACTCTAGGAGATTGCCCAAGGGAGTCAAGCATCTTAAGGACTTTCCAATCCTGCCTGGGGAGATCTTCAAGTACAAATGGACAGTGACAGTTGAGGATGGACCTACTAAGAGTGACCCCAGGTGTCTGACCAGATATTACTCTAGCTTTGTCAATATGGAAAGAGACCTGGCTTCAGGCTTGATAGGCCCTCTTCTCATCTGCTACAAAGAGTCTGTGGACCAGAGAGGAAATCAGATTATGTCTGACAAGAGAAATGTTATTCTGTTTTCAGTCTTTGATGAGAACAGAAGTTGGTACTTGACAGAGAACATTCAGAGATTTCTGCCTAACCCTGCAGGAGTGCAGCTTGAGGACCCTGAGTTTCAGGCTTCCAATATCATGCATTCTATCAATGGCTATGTGTTTGATAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTTGCCTATTGGTACATCCTGTCTATTGGGGCCCAAACTGATTTCTTGTCTGTGTTTTTCAGTGGATATACCTTTAAGCATAAGATGGTGTATGAAGATACCTTGACACTTTTCCCTTTCTCAGGAGAGACTGTCTTCATGTCTATGGAGAACCCTGGACTGTGGATCCTGGGATGCCACAACTCAGACTTCAGAAACAGGGGTATGACAGCTCTGCTTAAGGTCTCCTCATGTGACAAAAATACAGGAGACTACTATGAAGATTCCTATGAGGACATCTCTGCTTACCTGCTCAGCAAAAACAATGCCATTGAGCCCAGAAGCTTCAGCCAGAATTCCAGACATCCCAGTACTAGGCAGAAGCAGTTTAATGCCACCACCATCCCAGAAAATGACATTGAGAAGACAGACCCTTGGTTTGCCCATAGAACCCCAATGCCCAAGATTCAGAATGTGAGCAGCTCAGACTTGCTGATGCTGCTGAGGCAGTCTCCTACTCCCCATGGCCTGTCCCTCTCAGATCTGCAGGAAGCCAAGTATGAGACATTTTCTGATGACCCCTCCCCAGGGGCTATTGACAGTAACAACAGCCTGTCTGAAATGACCCACTTCAGACCTCAGCTCCACCACAGTGGGGATATGGTGTTTACCCCAGAGTCAGGCCTGCAGCTGAGACTGAATGAGAAGCTGGGAACCACAGCTGCCACAGAACTGAAGAAGCTGGATTTCAAGGTGAGCTCTACTTCAAATAATCTGATTTCCACTATCCCATCTGACAATTTGGCAGCTGGCACTGACAATACAAGCTCTCTGGGCCCACCTAGCATGCCAGTGCATTATGACAGCCAGTTGGACACCACTCTTTTTGGAAAGAAATCCAGTCCCCTGACAGAGTCAGGAGGACCCCTTTCTCTGTCTGAGGAGAACAATGATAGTAAGCTTCTGGAGTCAGGCTTGATGAACTCCCAGGAGAGTTCTTGGGGCAAGAATGTGAGCTCTAGGGAGATCACCAGAACCACTCTGCAGTCTGACCAAGAAGAGATTGATTATGATGACACTATCTCTGTTGAGATGAAGAAGGAGGACTTTGATATCTATGATGAGGATGAGAACCAGTCTCCAAGAAGTTTCCAGAAGAAGACCAGGCACTACTTTATTGCTGCTGTGGAGAGACTGTGGGACTATGGAATGTCCTCCTCCCCTCATGTCCTGAGAAATAGAGCTCAGTCAGGATCAGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAATTCACAGATGGAAGTTTTACCCAGCCACTGTATAGAGGTGAACTGAATGAACATCTGGGTCTGCTGGGGCCCTATATCAGGGCTGAGGTGGAGGACAATATTATGGTGACCTTTAGAAACCAGGCCTCAAGGCCCTACAGCTTCTACTCTTCCCTCATTTCATATGAGGAAGACCAGAGACAGGGAGCAGAGCCTAGAAAGAACTTTGTCAAGCCCAATGAAACCAAAACCTATTTCTGGAAGGTGCAGCACCACATGGCACCCACCAAAGATGAGTTTGACTGCAAAGCCTGGGCCTATTTCTCAGATGTGGATCTGGAGAAGGATGTCCATTCAGGTCTGATTGGACCACTCCTGGTGTGCCACACCAACACACTGAATCCAGCTCATGGCAGACAGGTGACAGTCCAGGAGTTTGCCCTGTTTTTCACTATTTTTGATGAGACCAAGAGCTGGTATTTTACAGAGAATATGGAAAGAAACTGCAGAGCCCCTTGTAATATCCAGATGGAGGACCCAACCTTTAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATTATGGATACCCTGCCTGGCCTGGTTATGGCCCAGGATCAGAGGATCAGGTGGTATCTGCTGTCAATGGGCTCCAATGAGAACATTCATAGTATTCACTTTTCAGGACATGTGTTCACAGTGAGAAAGAAGGAGGAGTATAAGATGGCCCTCTATAATCTCTACCCAGGGGTGTTTGAGACAGTTGAGATGCTGCCATCTAAGGCAGGCATCTGGAGAGTGGAATGCCTCATTGGAGAGCACCTGCATGCTGGCATGTCCACCCTGTTTCTGGTGTACTCCAACAAATGTCAGACCCCTCTTGGTATGGCCTCTGGCCACATTAGGGATTTCCAGATTACTGCCAGTGGTCAGTATGGGCAGTGGGCCCCTAAGCTGGCCAGACTGCACTATTCTGGGAGCATCAATGCCTGGTCCACCAAGGAACCTTTTTCTTGGATTAAAGTGGATCTGCTGGCCCCAATGATCATCCATGGGATCAAGACCCAGGGAGCTAGGCAGAAGTTCAGCAGCCTGTACATTAGTCAGTTCATCATTATGTACAGTCTGGATGGCAAAAAGTGGCAGACATACAGGGGCAATTCCACTGGAACCCTGATGGTGTTCTTTGGAAATGTGGATAGCAGTGGTATCAAGCACAACATTTTCAACCCTCCCATCATTGCCAGGTACATCAGACTGCATCCAACCCACTACAGCATTAGGAGCACCCTTAGGATGGAGCTCATGGGGTGTGACCTGAACTCTTGCTCTATGCCACTTGGAATGGAGAGCAAGGCCATCTCTGATGCCCAGATTACTGCTAGTTCCTACTTCACCAATATGTTTGCCACCTGGTCACCCAGCAAGGCCAGGCTGCACCTGCAGGGAAGGTCCAATGCTGGAGGCCTCAGGTGAACAATCCAAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACTATGAAGGTGACAGGAGTGACCACTCAGGGAGTGAAGAGCCTGCTGACCTCCATGTATGTGAAGGAGTTTCTGATCTCCAGCTCCCAGGATGGCCACCAGTGGACCCTGTTCTTTCAGAATGGCAAGGTGAAGGTGTTTCAGGGGAATCAGGACTCCTTCACCCCAGTGGTGAATTCCCTGGATCCCCCTCTGCTGACCAGATACCTGAGAATCCACCCCCAGTCTTGGGTGCACCAGATTGCCCTTAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA CpG-free human 5013 https://   0 396ATGCAAATTGAGTTAAGTACCTGTTTTTTCCTGTGCCTGTTGAGATTTTGCTTCTCTGCTACCAGAAGGTATTACCTGGGAhFVIII 226 www.ncbi.GCTGTGGAGCTGTCATGGGACTACATGCAGTCTGACCTGGGAGAGCTCCCTGTTGATGCTAGGTTTCCCCCTAGGGTGCVariant with nlm.nih.CAAAGTCCTTTCCTTTTAACACCTCAGTGGTGTACAAGAAGACACTGTTTGTGGAGTTTACTGACCACCTCTTCAATATTGF309S Mutation; gov/CTAAGCCAAGACCCCCTTGGATGGGTCTCTTGGGCCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTGATTACCCTCCOOL optimized pubmed/AAGAATATGGCTTCTCATCCTGTTAGTCTGCATGCAGTAGGGGTCAGCTATTGGAAGGCCAGTGAAGGAGCTGAGTAT14726380GATGACCAGACCAGCCAGAGAGAGAAAGAGGATGACAAGGTGTTCCCTGGGGGATCACACACCTATGTGTGGCAGGTGTTGAAGGAAAATGGACCCATGGCCTCTGACCCACTCTGCCTGACTTACTCCTACCTGTCTCATGTGGACCTGGTCAAAGATCTGAACTCTGGCCTGATTGGGGCACTGTTGGTGTGCAGAGAAGGGTCTCTTGCCAAGGAGAAGACTCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAAGGCAAGTCTTGGCATTCTGAGACTAAGAACAGCCTGATGCAAGATAGAGATGCTGCATCTGCCAGGGCCTGGCCTAAGATGCACACTGTGAATGGATATGTGAATAGGTCCTTGCCTGGCCTGATTGGATGCCATAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTTCACTCTATTTTTCTGGAGGGCCATACTTTCCTTGTGAGGAATCATAGACAGGCCTCTCTGGAGATTAGCCCTATTACATTTCTGACTGCTCAGACCCTCCTCATGGACTTGGGGCAGTTTCTGTTAAGCTGCCACATCAGCAGCCATCAACATGATGGTATGGAAGCCTATGTCAAAGTGGATAGCTGCCCTGAAGAACCACAGCTTAGGATGAAGAACAATGAGGAGGCAGAGGACTATGATGATGATCTGACTGATTCTGAAATGGATGTGGTGAGATTTGATGATGATAATAGCCCTTCCTTTATCCAGATCAGGTCAGTGGCCAAGAAGCACCCAAAGACCTGGGTGCATTATATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCTCTGGTGCTGGCCCCTGATGATAGAAGCTATAAGTCTCAATACTTGAATAATGGCCCACAGAGGATTGGGAGAAAGTACAAAAAGGTGAGATTTATGGCCTACACTGATGAGACTTTCAAGACCAGAGAGGCCATCCAGCATGAATCAGGCATTTTGGGTCCCCTGCTGTATGGAGAGGTGGGGGATACCCTGCTGATCATCTTTAAGAACCAGGCCAGCAGACCTTACAACATTTACCCTCATGGGATCACAGATGTGAGGCCACTCTACTCTAGGAGATTGCCCAAGGGAGTCAAGCATCTTAAGGACTTTCCAATCCTGCCTGGGGAGATCTTCAAGTACAAATGGACAGTGACAGTTGAGGATGGACCTACTAAGAGTGACCCCAGGTGTCTGACCAGATATTACTCTAGCTTTGTCAATATGGAAAGAGACCTGGCTTCAGGCTTGATAGGCCCTCTTCTCATCTGCTACAAAGAGTCTGTGGACCAGAGAGGAAATCAGATTATGTCTGACAAGAGAAATGTTATTCTGTTTTCAGTCTTTGATGAGAACAGAAGTTGGTACTTGACAGAGAACATTCAGAGATTTCTGCCTAACCCTGCAGGAGTGCAGCTTGAGGACCCTGAGTTTCAGGCTTCCAATATCATGCATTCTATCAATGGCTATGTGTTTGATAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTTGCCTATTGGTACATCCTGTCTATTGGGGCCCAAACTGATTTCTTGTCTGTGTTTTTCAGTGGATATACCTTTAAGCATAAGATGGTGTATGAAGATACCTTGACACTTTTCCCTTTCTCAGGAGAGACTGTCTTCATGTCTATGGAGAACCCTGGACTGTGGATCCTGGGATGCCACAACTCAGACTTCAGAAACAGGGGTATGACAGCTCTGCTTAAGGTCTCCTCATGTGACAAAAATACAGGAGACTACTATGAAGATTCCTATGAGGACATCTCTGCTTACCTGCTCAGCAAAAACAATGCCATTGAGCCCAGAAGCTTCAGCCAGAATTCCAGACATCCCAGTACTAGGCAGAAGCAGTTTAATGCCACCACCATCCCAGAAAATGACATTGAGAAGACAGACCCTTGGTTTGCCCATAGAACCCCAATGCCCAAGATTCAGAATGTGAGCAGCTCAGACTTGCTGATGCTGCTGAGGCAGTCTCCTACTCCCCATGGCCTGTCCCTCTCAGATCTGCAGGAAGCCAAGTATGAGACATTTTCTGATGACCCCTCCCCAGGGGCTATTGACAGTAACAACAGCCTGTCTGAAATGACCCACTTCAGACCTCAGCTCCACCACAGTGGGGATATGGTGTTTACCCCAGAGTCAGGCCTGCAGCTGAGACTGAATGAGAAGCTGGGAACCACAGCTGCCACAGAACTGAAGAAGCTGGATTTCAAGGTGAGCTCTACTTCAAATAATCTGATTTCCACTATCCCATCTGACAATTTGGCAGCTGGCACTGACAATACAAGCTCTCTGGGCCCACCTAGCATGCCAGTGCATTATGACAGCCAGTTGGACACCACTCTTTTTGGAAAGAAATCCAGTCCCCTGACAGAGTCAGGAGGACCCCTTTCTCTGTCTGAGGAGAACAATGATAGTAAGCTTCTGGAGTCAGGCTTGATGAACTCCCAGGAGAGTTCTTGGGGCAAGAATGTGAGCTCTAGGGAGATCACCAGAACCACTCTGCAGTCTGACCAAGAAGAGATTGATTATGATGACACTATCTCTGTTGAGATGAAGAAGGAGGACTTTGATATCTATGATGAGGATGAGAACCAGTCTCCAAGAAGTTTCCAGAAGAAGACCAGGCACTACTTTATTGCTGCTGTGGAGAGACTGTGGGACTATGGAATGTCCTCCTCCCCTCATGTCCTGAGAAATAGAGCTCAGTCAGGATCAGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAATTCACAGATGGAAGTTTTACCCAGCCACTGTATAGAGGTGAACTGAATGAACATCTGGGTCTGCTGGGGCCCTATATCAGGGCTGAGGTGGAGGACAATATTATGGTGACCTTTAGAAACCAGGCCTCAAGGCCCTACAGCTTCTACTCTTCCCTCATTTCATATGAGGAAGACCAGAGACAGGGAGCAGAGCCTAGAAAGAACTTTGTCAAGCCCAATGAAACCAAAACCTATTTCTGGAAGGTGCAGCACCACATGGCACCCACCAAAGATGAGTTTGACTGCAAAGCCTGGGCCTATTTCTCAGATGTGGATCTGGAGAAGGATGTCCATTCAGGTCTGATTGGACCACTCCTGGTGTGCCACACCAACACACTGAATCCAGCTCATGGCAGACAGGTGACAGTCCAGGAGTTTGCCCTGTTTTTCACTATTTTTGATGAGACCAAGAGCTGGTATTTTACAGAGAATATGGAAAGAAACTGCAGAGCCCCTTGTAATATCCAGATGGAGGACCCAACCTTTAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATTATGGATACCCTGCCTGGCCTGGTTATGGCCCAGGATCAGAGGATCAGGTGGTATCTGCTGTCAATGGGCTCCAATGAGAACATTCATAGTATTCACTTTTCAGGACATGTGTTCACAGTGAGAAAGAAGGAGGAGTATAAGATGGCCCTCTATAATCTCTACCCAGGGGTGTTTGAGACAGTTGAGATGCTGCCATCTAAGGCAGGCATCTGGAGAGTGGAATGCCTCATTGGAGAGCACCTGCATGCTGGCATGTCCACCCTGTTTCTGGTGTACTCCAACAAATGTCAGACCCCTCTTGGTATGGCCTCTGGCCACATTAGGGATTTCCAGATTACTGCCAGTGGTCAGTATGGGCAGTGGGCCCCTAAGCTGGCCAGACTGCACTATTCTGGGAGCATCAATGCCTGGTCCACCAAGGAACCTTTTTCTTGGATTAAAGTGGATCTGCTGGCCCCAATGATCATCCATGGGATCAAGACCCAGGGAGCTAGGCAGAAGTTCAGCAGCCTGTACATTAGTCAGTTCATCATTATGTACAGTCTGGATGGCAAAAAGTGGCAGACATACAGGGGCAATTCCACTGGAACCCTGATGGTGTTCTTTGGAAATGTGGATAGCAGTGGTATCAAGCACAACATTTTCAACCCTCCCATCATTGCCAGGTACATCAGACTGCATCCAACCCACTACAGCATTAGGAGCACCCTTAGGATGGAGCTCATGGGGTGTGACCTGAACTCTTGCTCTATGCCACTTGGAATGGAGAGCAAGGCCATCTCTGATGCCCAGATTACTGCTAGTTCCTACTTCACCAATATGTTTGCCACCTGGTCACCCAGCAAGGCCAGGCTGCACCTGCAGGGAAGGTCCAATGCTGGAGGCCTCAGGTGAACAATCCAAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACTATGAAGGTGACAGGAGTGACCACTCAGGGAGTGAAGAGCCTGCTGACCTCCATGTATGTGAAGGAGTTTCTGATCTCCAGCTCCCAGGATGGCCACCAGTGGACCCTGTTCTTTCAGAATGGCAAGGTGAAGGTGTTTCAGGGGAATCAGGACTCCTTCACCCCAGTGGTGAATTCCCTGGATCCCCCTCTGCTGACCAGATACCTGAGAATCCACCCCCAGTCTTGGGTGCACCAGATTGCCCTTAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA CpG-free human 397MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPhFVIII 226PWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGVariant withPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWF309S Mutation;PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLSCHICOOL optimized-SSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDamino acidWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPsequenceHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPANGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVITQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY-

(iii) FVIII Therapeutic Proteins and Uses Thereof for the Treatment ofHemophilia A

The ceDNA vectors described herein can be used to deliver therapeuticFVIII proteins for treatment of hemophilia A associated withinappropriate expression of the FVIII protein and/or mutations withinthe FVIII proteins.

ceDNA vectors as described herein can be used to express any desiredFVIII therapeutic protein. Exemplary therapeutic FVIII therapeuticproteins include but are not limited to any FVIII protein expressed bythe sequences as set forth in Table 1 herein.

In one embodiment, the expressed FVIII therapeutic protein is functionalfor the treatment of a Hemophilia A. In some embodiments, FVIIItherapeutic protein does not cause an immune system reaction.

In another embodiment, the ceDNA vectors encoding FVIII therapeuticprotein or fragment thereof (e.g., functional fragment) can be used togenerate a chimeric protein. Thus, it is specifically contemplatedherein that a ceDNA vector expressing a chimeric protein can beadministered to e.g., to any one or more tissues selected from: liver,kidneys, gallbladder, prostate, adrenal gland. In some embodiments, whena ceDNA vector expressing FVIII is administered to an infant, oradministered to a subject in utero, one can administer a ceDNA vectorexpressing FVIII to any one or more tissues selected from: liver,adrenal gland, heart, intestine, lung, and stomach, or to a liver stemcell precursor thereof for the in vivo or ex vivo treatment ofhemophilia A.

Hemophilia:

Hemophilia A is a genetic deficiency in clotting factor VIII, whichcauses increased bleeding and usually affects males. In the majority ofcases it is inherited as an X-linked recessive trait, though there arecases which arise from spontaneous mutations. In terms of the symptomsof hemophilia A, there are internal or external bleeding episodes.Individuals with more severe hemophilia suffer more severe and morefrequent bleeding, while others with mild hemophilia typically suffermore minor symptoms except after surgery or serious trauma. Moderatehemophiliacs have variable symptoms which manifest along a spectrumbetween severe and mild forms.

Current treatments to prevent bleeding in people with hemophilia Ainvolves Factor VIII medication. Most individuals with severe hemophiliarequire regular supplementation with intravenous recombinant or plasmaconcentrate Factor VIII. Recombinant blood clotting factor VIII is oneof the most complex proteins for industrial manufacturing due to the lowefficiency of its gene transcription, massive intracellular loss of itsproprotein during post-translational processing, and the instability ofthe secreted protein. Mild hemophiliacs can manage their condition withdesmopressin, a drug which releases stored factor VIII from blood vesselwalls.

There are many complications related to treatment of hemophilia A. Inchildren, an easily accessible intravenous port can be inserted tominimize frequent traumatic intravenous cannulation. However, theseports are associated with high infection rate and a risk of clotsforming at the tip of the catheter, rendering it useless. Viralinfections can be common in hemophiliacs due to frequent bloodtransfusions which put patients at risk of acquiring blood borneinfections, such as HIV, hepatitis B and hepatitis C. Prion infectionscan also be transmitted by blood transfusions. Another therapeuticcomplication of hemophilia A is the development of inhibitor antibodiesagainst factor VIII due to frequent infusions. These develop as the bodyrecognizes the infused factor VIII as foreign, as the body does notproduce its own copy. In these individuals, activated factor VII, aprecursor to factor VIII in the coagulation cascade, can be infused as atreatment for hemorrhage in individuals with hemophilia and antibodiesagainst replacement factor VIII.

Coagulation Cascade

Coagulation, also known as clotting, is the process by which bloodchanges from a liquid to a gel, forming a blood clot. It potentiallyresults in hemostasis, the cessation of blood loss from a damagedvessel, followed by repair. The mechanism of coagulation involvesactivation, adhesion and aggregation of platelets along with depositionand maturation of fibrin. Disorders of coagulation are disease stateswhich can result in bleeding (hemorrhage or bruising) or obstructiveclotting (thrombosis).

Coagulation begins almost instantly after an injury to the blood vesselhas damaged the endothelium lining the blood vessel. Exposure of bloodto the subendothelial space initiates two processes: changes inplatelets, and the exposure of subendothelial tissue factor to plasmaFactor VII, which ultimately leads to fibrin formation. Plateletsimmediately form a plug at the site of injury; this is called primaryhemostasis. Secondary hemostasis occurs simultaneously: additionalcoagulation factors or clotting factors beyond Factor VII (includingFactor VIII) respond in a complex cascade to form fibrin strands, whichstrengthen the platelet plug.

The coagulation cascade of secondary hemostasis has two initial pathwayswhich lead to fibrin formation. These are the contact activation pathway(also known as the intrinsic pathway), and the tissue factor pathway(also known as the extrinsic pathway), which both lead to the samefundamental reactions that produce fibrin. The primary pathway for theinitiation of blood coagulation is the tissue factor (extrinsic)pathway. The pathways are a series of reactions, in which a zymogen(inactive enzyme precursor) of a serine protease and its glycoproteinco-factor are activated to become active components that then catalyzethe next reaction in the cascade, ultimately resulting in cross-linkedfibrin. Coagulation factors are generally indicated by Roman numerals,with a lowercase a appended to indicate an active form.

The coagulation factors are generally serine proteases (enzymes), whichact by cleaving downstream proteins. The exceptions are tissue factor,FV, FVIII, FXIII. Tissue factor, FV and FVIII are glycoproteins, andFactor XIII is a transglutaminase. The coagulation factors circulate asinactive zymogens. The coagulation cascade is therefore classicallydivided into three pathways. The tissue factor and contact activationpathways both activate the “final common pathway” of factor X, thrombinand fibrin.

The main role of the tissue factor (extrinsic) pathway is to generate a“thrombin burst”, a process by which thrombin, the most importantconstituent of the coagulation cascade in terms of its feedbackactivation roles, is released very rapidly. FVIIa circulates in a higheramount than any other activated coagulation factor. The process includesthe following steps:

Step 1: Following damage to the blood vessel, FVII leaves thecirculation and comes into contact with tissue factor (TF) expressed ontissue-factor-bearing cells (stromal fibroblasts and leukocytes),forming an activated complex (TF-FVIIa).

Step 2: TF-FVIIa activates FIX and FX.

Step 3: FVII is itself activated by thrombin, FXIa, FXII and FXa.

Step 4: The activation of FX (to form FXa) by TF-FVIIa is almostimmediately inhibited by tissue factor pathway inhibitor (TFPI).

Step 5: FXa and its co-factor FVa form the prothrombinase complex, whichactivates prothrombin to thrombin.

Step 6: Thrombin then activates other components of the coagulationcascade, including FV and FVIII (which forms a complex with FIX), andactivates and releases FVIII from being bound to von Willebrand factor(vWF).

Step 7: FVIIIa is the co-factor of FIXa, and together they form the“tenase” complex, which activates FX; and so the cycle continues.

The contact activation (intrinsic) pathway begins with formation of theprimary complex on collagen by high-molecular-weight kininogen (HMWK),prekallikrein, and FXII (Hageman factor). Prekallikrein is converted tokallikrein and FXII becomes FXIIa. FXIIa converts FXI into FXIa. FactorXIa activates FIX, which with its co-factor FVIIIa form the tenasecomplex, which activates FX to FXa. The minor role that the contactactivation pathway has in initiating clot formation can be illustratedby the fact that patients with severe deficiencies of FXII, HMWK, andprekallikrein do not have a bleeding disorder. Instead, contactactivation system is more involved in inflammation, and innate immunity.

The final common pathway shared by the intrinsic and extrinsiccoagulation pathways involves the conversion of prothrombin intothrombin and fibrinogen into fibrin. Thrombin has a large array offunctions, not only the conversion of fibrinogen to fibrin, the buildingblock of a hemostatic plug. In addition, it is the most importantplatelet activator and on top of that it activates Factors VIII and Vand their inhibitor protein C (in the presence of thrombomodulin), andit activates Factor XIII, which forms covalent bonds that crosslink thefibrin polymers that form from activated monomers.

Following activation by the contact factor or tissue factor pathways,the coagulation cascade is maintained in a prothrombotic state by thecontinued activation of FVIII and FIX to form the tenase complex, untilit is down-regulated by the anticoagulant pathways. The methods compriseadministering to the subject an effective amount of a compositioncomprising a ceDNA vector encoding the FVIII therapeutic protein orfragment thereof (e.g., functional fragment) as described herein. Aswill be appreciated by a skilled practitioner, the term “effectiveamount” refers to the amount of the ceDNA composition administered thatresults in expression of the protein in a “therapeutically effectiveamount” for the treatment of a disease or disorder.

The dosage ranges for the composition comprising a ceDNA vector encodingthe FVIII therapeutic protein or fragment thereof (e.g., functionalfragment) depends upon the potency (e.g., efficiency of the promoter),and includes amounts large enough to produce the desired effect, e.g.,expression of the desired FVIII therapeutic protein, for the treatmentof Phenylketonuria (hemophilia A). The dosage should not be so large asto cause unacceptable adverse side effects. Generally, the dosage willvary with the particular characteristics of the ceDNA vector, expressionefficiency and with the age, condition, and sex of the patient. Thedosage can be determined by one of skill in the art and, unliketraditional AAV vectors, can also be adjusted by the individualphysician in the event of any complication because ceDNA vectors do notcomprise immune activating capsid proteins that prevent repeat dosing.

Administration of the ceDNA compositions described herein can berepeated for a limited period of time. In some embodiments, the dosesare given periodically or by pulsed administration. In a preferredembodiment, the doses recited above are administered over severalmonths. The duration of treatment depends upon the subject's clinicalprogress and responsiveness to therapy. Booster treatments over time arecontemplated. Further, the level of expression can be titrated as thesubject grows.

An FVIII therapeutic protein can be expressed in a subject for at least1 week, at least 2 weeks, at least 1 month, at least 2 months, at least6 months, at least 12 months/one year, at least 2 years, at least 5years, at least 10 years, at least 15 years, at least 20 years, at least30 years, at least 40 years, at least 50 years or more. Long-termexpression can be achieved by repeated administration of the ceDNAvectors described herein at predetermined or desired intervals.

As used herein, the term “therapeutically effective amount” is an amountof an expressed FVIII therapeutic protein, or functional fragmentthereof that is sufficient to produce a statistically significant,measurable change in expression of a disease biomarker or reduction in agiven disease symptom (see “Efficacy Measurement” below). Such effectiveamounts can be gauged in clinical trials as well as animal studies for agiven ceDNA composition.

Precise amounts of the ceDNA vector required to be administered dependon the judgment of the practitioner and are particular to eachindividual. Suitable regimes for administration are also variable, butare typified by an initial administration followed by repeated doses atone or more intervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated, particularly for the treatment of acutediseases/disorders.

Agents useful in the methods and compositions described herein can beadministered topically, intravenously (by bolus or continuous infusion),intracellular injection, intratissue injection, orally, by inhalation,intraperitoneally, intramuscularly, subcutaneously, intracavity, and canbe delivered by peristaltic means, if desired, or by other means knownby those skilled in the art. The agent can be administered systemically,if so desired. It can also be administered in utero.

The efficacy of a given treatment for hemophilia A, can be determined bythe skilled clinician. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of the disease or disorder is/are altered in a beneficialmanner, or other clinically accepted symptoms or markers of disease areimproved, or ameliorated, e.g., by at least 10% following treatment witha ceDNA vector encoding FVIII, or a functional fragment thereof.Efficacy can also be measured by failure of an individual to worsen asassessed by stabilization of the disease, or the need for medicalinterventions (i.e., progression of the disease is halted or at leastslowed). Methods of measuring these indicators are known to those ofskill in the art and/or described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human, or a mammal) and includes: (1) inhibiting thedisease, e.g., arresting, or slowing progression of the disease ordisorder; or (2) relieving the disease, e.g., causing regression ofsymptoms; and (3) preventing or reducing the likelihood of thedevelopment of the disease, or preventing secondary diseases/disordersassociated with the disease, such as liver or kidney failure. Aneffective amount for the treatment of a disease means that amount which,when administered to a mammal in need thereof, is sufficient to resultin effective treatment as that term is defined herein, for that disease.

Efficacy of an agent can be determined by assessing physical indicatorsthat are particular to hemophilia A. Standard methods of analysis ofhemophilia A indicators are known in the art.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein can also encode co-factors or other polypeptides, senseor antisense oligonucleotides, or RNAs (coding or non-coding; e.g.,siRNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g.,antagoMiR)) that can be used in conjunction with the FVIII proteinexpressed from the ceDNA. Additionally, expression cassettes comprisingsequence encoding an FVIII protein can also include an exogenoussequence that encodes a reporter protein to be used for experimental ordiagnostic purposes, such as β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art.

In one embodiment, the ceDNA vector comprises a nucleic acid sequence toexpress the FVIII protein that is functional for the treatment ofhemophilia A. In a preferred embodiment, the therapeutic FVIII proteindoes not cause an immune system reaction, unless so desired.

III. ceDNA Vector in General for Use in Production of FVIII TherapeuticProteins

Embodiments of the invention are based on methods and compositionscomprising close ended linear duplexed (ceDNA) vectors that can expressthe FVIII transgene. In some embodiments, the transgene is a sequenceencoding an FVIII protein. The ceDNA vectors for expression of FVIIIprotein as described herein are not limited by size, thereby permitting,for example, expression of all of the components necessary forexpression of a transgene from a single vector. The ceDNA vector forexpression of FVIII protein is preferably duplex, e.g.self-complementary, over at least a portion of the molecule, such as theexpression cassette (e.g. ceDNA is not a double stranded circularmolecule). The ceDNA vector has covalently closed ends, and thus isresistant to exonuclease digestion (e.g. exonuclease I or exonucleaseIII), e.g. for over an hour at 37° C.

In general, a ceDNA vector for expression of FVIII protein as disclosedherein, comprises in the 5′ to 3′ direction: a first adeno-associatedvirus (AAV) inverted terminal repeat (ITR), a nucleotide sequence ofinterest (for example an expression cassette as described herein) and asecond AAV ITR. The ITR sequences selected from any of: (i) at least oneWT ITR and at least one modified AAV inverted terminal repeat (mod-ITR)(e.g., asymmetric modified ITRs); (ii) two modified ITRs where themod-ITR pair have a different three-dimensional spatial organizationwith respect to each other (e.g., asymmetric modified ITRs), or (iii)symmetrical or substantially symmetrical WT-WT ITR pair, where eachWT-ITR has the same three-dimensional spatial organization, or (iv)symmetrical or substantially symmetrical modified ITR pair, where eachmod-ITR has the same three-dimensional spatial organization.

Encompassed herein are methods and compositions comprising the ceDNAvector for FVIII protein production, which may further include adelivery system, such as but not limited to, a liposome nanoparticledelivery system. Non-limiting exemplary liposome nanoparticle systemsencompassed for use are disclosed herein. In some aspects, thedisclosure provides for a lipid nanoparticle comprising ceDNA and anionizable lipid. For example, a lipid nanoparticle formulation that ismade and loaded with a ceDNA vector obtained by the process is disclosedin International Application PCT/US2018/050042, filed on Sep. 7, 2018,which is incorporated herein.

The ceDNA vectors for expression of FVIII protein as disclosed hereinhave no packaging constraints imposed by the limiting space within theviral capsid. ceDNA vectors represent a viable eukaryotically-producedalternative to prokaryote-produced plasmid DNA vectors, as opposed toencapsulated AAV genomes. This permits the insertion of controlelements, e.g., regulatory switches as disclosed herein, largetransgenes, multiple transgenes etc.

FIG. 1A-1E show schematics of non-limiting, exemplary ceDNA vectors forexpression of FVIII protein, or the corresponding sequence of ceDNAplasmids. ceDNA vectors for expression of FVIII protein are capsid-freeand can be obtained from a plasmid encoding in this order: a first ITR,an expression cassette comprising a transgene and a second ITR. Theexpression cassette may include one or more regulatory sequences thatallows and/or controls the expression of the transgene, e.g., where theexpression cassette can comprise one or more of, in this order: anenhancer/promoter, an ORF reporter (transgene), a post-transcriptionregulatory element (e.g., WPRE), and a polyadenylation and terminationsignal (e.g., BGH polyA).

The expression cassette can also comprise an internal ribosome entrysite (IRES) and/or a 2A element. The cis-regulatory elements include,but are not limited to, a promoter, a riboswitch, an insulator, amir-regulatable element, a post-transcriptional regulatory element, atissue- and cell type-specific promoter and an enhancer. In someembodiments the ITR can act as the promoter for the transgene, e.g.,FVIII protein. In some embodiments, the ceDNA vector comprisesadditional components to regulate expression of the transgene, forexample, a regulatory switch, which are described herein in the sectionentitled “Regulatory Switches” for controlling and regulating theexpression of the FVIII protein, and can include if desired, aregulatory switch which is a kill switch to enable controlled cell deathof a cell comprising a ceDNA vector.

The expression cassette can comprise more than 4000 nucleotides, 5000nucleotides, 10,000 nucleotides or 20,000 nucleotides, or 30,000nucleotides, or 40,000 nucleotides or 50,000 nucleotides, or any rangebetween about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, ormore than 50,000 nucleotides. In some embodiments, the expressioncassette can comprise a transgene in the range of 500 to 50,000nucleotides in length. In some embodiments, the expression cassette cancomprise a transgene in the range of 500 to 75,000 nucleotides inlength. In some embodiments, the expression cassette can comprise atransgene which is in the range of 500 to 10,000 nucleotides in length.In some embodiments, the expression cassette can comprise a transgenewhich is in the range of 1000 to 10,000 nucleotides in length. In someembodiments, the expression cassette can comprise a transgene which isin the range of 500 to 5,000 nucleotides in length. The ceDNA vectors donot have the size limitations of encapsidated AAV vectors, thus enabledelivery of a large-size expression cassette to provide efficienttransgene expression. In some embodiments, the ceDNA vector is devoid ofprokaryote-specific methylation.

ceDNA expression cassette can include, for example, an expressibleexogenous sequence (e.g., open reading frame) or transgene that encodesa protein that is either absent, inactive, or insufficient activity inthe recipient subject or a gene that encodes a protein having a desiredbiological or a therapeutic effect. The transgene can encode a geneproduct that can function to correct the expression of a defective geneor transcript. In principle, the expression cassette can include anygene that encodes a protein, polypeptide or RNA that is either reducedor absent due to a mutation or which conveys a therapeutic benefit whenoverexpressed is considered to be within the scope of the disclosure.

The expression cassette can comprise any transgene (e.g., encoding FVIIIprotein), for example, FVIII protein useful for treating hemophilia A ina subject, i.e., a therapeutic FVIII protein. A ceDNA vector can be usedto deliver and express any FVIII protein of interest in the subject,alone or in combination with nucleic acids encoding polypeptides, ornon-coding nucleic acids (e.g., RNAi, miRs etc.), as well as exogenousgenes and nucleotide sequences, including virus sequences in a subjects'genome, e.g., HIV virus sequences and the like. Preferably a ceDNAvector disclosed herein is used for therapeutic purposes (e.g., formedical, diagnostic, or veterinary uses) or immunogenic polypeptides. Incertain embodiments, a ceDNA vector is useful to express any gene ofinterest in the subject, which includes one or more polypeptides,peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisenseoligonucleotides, antisense polynucleotides, or RNAs (coding ornon-coding; e.g., siRNAs, shRNAs, micro-RNAs, and their antisensecounterparts (e.g., antagoMiR)), antibodies, fusion proteins, or anycombination thereof.

The expression cassette can also encode polypeptides, sense or antisenseoligonucleotides, or RNAs (coding or non-coding; e.g., siRNAs, shRNAs,micro-RNAs, and their antisense counterparts (e.g., antagoMiR)).Expression cassettes can include an exogenous sequence that encodes areporter protein to be used for experimental or diagnostic purposes,such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase,thymidine kinase, green fluorescent protein (GFP), chloramphenicolacetyltransferase (CAT), luciferase, and others well known in the art.

Sequences provided in the expression cassette, expression construct of aceDNA vector for expression of FVIII protein described herein can becodon optimized for the target host cell. As used herein, the term“codon optimized” or “codon optimization” refers to the process ofmodifying a nucleic acid sequence for enhanced expression in the cellsof the vertebrate of interest, e.g., mouse or human, by replacing atleast one, more than one, or a significant number of codons of thenative sequence (e.g., a prokaryotic sequence) with codons that are morefrequently or most frequently used in the genes of that vertebrate.Various species exhibit particular bias for certain codons of aparticular amino acid. Typically, codon optimization does not alter theamino acid sequence of the original translated protein. Optimized codonscan be determined using e.g., Aptagen's Gene Forge® codon optimizationand custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd.Suite 300, Herndon, Va. 20171) or another publicly available database.In some embodiments, the nucleic acid encoding the FVIII protein isoptimized for human expression, and/or is a human FVIII, or functionalfragment thereof, as known in the art.

A transgene expressed by the ceDNA vector for expression of FVIIIprotein as disclosed herein encodes FVIII protein. There are manystructural features of ceDNA vectors for expression of FVIII proteinthat differ from plasmid-based expression vectors. ceDNA vectors maypossess one or more of the following features: the lack of original(i.e. not inserted) bacterial DNA, the lack of a prokaryotic origin ofreplication, being self-containing, i.e., they do not require anysequences other than the two ITRs, including the Rep binding andterminal resolution sites (RBS and TRS), and an exogenous sequencebetween the ITRs, the presence of ITR sequences that form hairpins, andthe absence of bacterial-type DNA methylation or indeed any othermethylation considered abnormal by a mammalian host. In general, it ispreferred for the present vectors not to contain any prokaryotic DNA butit is contemplated that some prokaryotic DNA may be inserted as anexogenous sequence, as a non-limiting example in a promoter or enhancerregion. Another important feature distinguishing ceDNA vectors fromplasmid expression vectors is that ceDNA vectors are single-strandlinear DNA having closed ends, while plasmids are always double-strandDNA.

ceDNA vectors for expression of FVIII protein produced by the methodsprovided herein preferably have a linear and continuous structure ratherthan a non-continuous structure, as determined by restriction enzymedigestion assay (FIG. 4D). The linear and continuous structure isbelieved to be more stable from attack by cellular endonucleases, aswell as less likely to be recombined and cause mutagenesis. Thus, aceDNA vector in the linear and continuous structure is a preferredembodiment. The continuous, linear, single strand intramolecular duplexceDNA vector can have covalently bound terminal ends, without sequencesencoding AAV capsid proteins. These ceDNA vectors are structurallydistinct from plasmids (including ceDNA plasmids described herein),which are circular duplex nucleic acid molecules of bacterial origin.The complimentary strands of plasmids may be separated followingdenaturation to produce two nucleic acid molecules, whereas in contrast,ceDNA vectors, while having complimentary strands, are a single DNAmolecule and therefore even if denatured, remain a single molecule. Insome embodiments, ceDNA vectors as described herein can be producedwithout DNA base methylation of prokaryotic type, unlike plasmids.Therefore, the ceDNA vectors and ceDNA-plasmids are different both interm of structure (in particular, linear versus circular) and also inview of the methods used for producing and purifying these differentobjects (see below), and also in view of their DNA methylation which isof prokaryotic type for ceDNA-plasmids and of eukaryotic type for theceDNA vector.

There are several advantages of using a ceDNA vector for expression ofFVIII protein as described herein over plasmid-based expression vectors,such advantages include, but are not limited to: 1) plasmids containbacterial DNA sequences and are subjected to prokaryotic-specificmethylation, e.g., 6-methyl adenosine and 5-methyl cytosine methylation,whereas capsid-free AAV vector sequences are of eukaryotic origin and donot undergo prokaryotic-specific methylation; as a result, capsid-freeAAV vectors are less likely to induce inflammatory and immune responsescompared to plasmids; 2) while plasmids require the presence of aresistance gene during the production process, ceDNA vectors do not; 3)while a circular plasmid is not delivered to the nucleus uponintroduction into a cell and requires overloading to bypass degradationby cellular nucleases, ceDNA vectors contain viral cis-elements, i.e.,ITRs, that confer resistance to nucleases and can be designed to betargeted and delivered to the nucleus. It is hypothesized that theminimal defining elements indispensable for ITR function are aRep-binding site (RBS; 5′-GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60) for AAV2)and a terminal resolution site (TRS; 5′-AGTTGG-3′ (SEQ ID NO: 64) forAAV2) plus a variable palindromic sequence allowing for hairpinformation; and 4) ceDNA vectors do not have the over-representation ofCpG dinucleotides often found in prokaryote-derived plasmids thatreportedly binds a member of the Toll-like family of receptors,eliciting a T cell-mediated immune response. In contrast, transductionswith capsid-free AAV vectors disclosed herein can efficiently targetcell and tissue-types that are difficult to transduce with conventionalAAV virions using various delivery reagent.

IV. Inverted Terminal Repeats (ITRs)

As disclosed herein, ceDNA vectors for expression of FVIII proteincontain a transgene or heterologous nucleic acid sequence positionedbetween two inverted terminal repeat (ITR) sequences, where the ITRsequences can be an asymmetrical ITR pair or a symmetrical- orsubstantially symmetrical ITR pair, as these terms are defined herein. AceDNA vector as disclosed herein can comprise ITR sequences that areselected from any of: (i) at least one WT ITR and at least one modifiedAAV inverted terminal repeat (mod-ITR) (e.g., asymmetric modified ITRs);(ii) two modified ITRs where the mod-ITR pair have a differentthree-dimensional spatial organization with respect to each other (e.g.,asymmetric modified ITRs), or (iii) symmetrical or substantiallysymmetrical WT-WT ITR pair, where each WT-ITR has the samethree-dimensional spatial organization, or (iv) symmetrical orsubstantially symmetrical modified ITR pair, where each mod-ITR has thesame three-dimensional spatial organization, where the methods of thepresent disclosure may further include a delivery system, such as butnot limited to a liposome nanoparticle delivery system.

In some embodiments, the ITR sequence can be from viruses of theParvoviridae family, which includes two subfamilies: Parvovirinae, whichinfect vertebrates, and Densovirinae, which infect insects. Thesubfamily Parvovirinae (referred to as the parvoviruses) includes thegenus Dependovirus, the members of which, under most conditions, requirecoinfection with a helper virus such as adenovirus or herpes virus forproductive infection. The genus Dependovirus includes adeno-associatedvirus (AAV), which normally infects humans (e.g., serotypes 2, 3A, 3B,5, and 6) or primates (e.g., serotypes 1 and 4), and related virusesthat infect other warm-blooded animals (e.g., bovine, canine, equine,and ovine adeno-associated viruses). The parvoviruses and other membersof the Parvoviridae family are generally described in Kenneth I. Berns,“Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FIELDSVIROLOGY (3d Ed. 1996).

While ITRs exemplified in the specification and Examples herein are AAV2WT-ITRs, one of ordinary skill in the art is aware that one can asstated above use ITRs from any known parvovirus, for example adependovirus such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5,AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, andAAV-DJ8 genome. E.g., NCBI: NC 002077; NC 001401; NC001729; NC001829;NC006152; NC 006260; NC 006261), chimeric ITRs, or ITRs from anysynthetic AAV. In some embodiments, the AAV can infect warm-bloodedanimals, e.g., avian (AAAV), bovine (BAAV), canine, equine, and ovineadeno-associated viruses. In some embodiments the ITR is from B19parvovirus (GenBank Accession No: NC 000883), Minute Virus from Mouse(MVM) (GenBank Accession No. NC 001510); goose parvovirus (GenBankAccession No. NC 001701); snake parvovirus 1 (GenBank Accession No. NC006148). In some embodiments, the 5′ WT-ITR can be from one serotype andthe 3′ WT-ITR from a different serotype, as discussed herein.

An ordinarily skilled artisan is aware that ITR sequences have a commonstructure of a double-stranded Holliday junction, which typically is aT-shaped or Y-shaped hairpin structure (see e.g., FIG. 2A and FIG. 3A),where each WT-ITR is formed by two palindromic arms or loops (B-B′ andC-C′) embedded in a larger palindromic arm (A-A′), and a single strandedD sequence, (where the order of these palindromic sequences defines theflip or flop orientation of the ITR). See, for example, structuralanalysis and sequence comparison of ITRs from different AAV serotypes(AAV1-AAV6) and described in Grimm et al., J. Virology, 2006; 80(1);426-439; Yan et al., J. Virology, 2005; 364-379; Duan et al., Virology1999; 261; 8-14. One of ordinary skill in the art can readily determineWT-ITR sequences from any AAV serotype for use in a ceDNA vector orceDNA-plasmid based on the exemplary AAV2 ITR sequences provided herein.See, for example, the sequence comparison of ITRs from different AAVserotypes (AAV1-AAV6, and avian AAV (AAAV) and bovine AAV (BAAV))described in Grimm et al., J. Virology, 2006; 80(1); 426-439; that showthe % identity of the left ITR of AAV2 to the left ITR from otherserotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6(left ITR) (100%) and AAV-6 (right ITR) (82%).

A. Symmetrical ITR Pairs

In some embodiments, a ceDNA vector for expression of FVIII protein asdescribed herein comprises, in the 5′ to 3′ direction: a firstadeno-associated virus (AAV) inverted terminal repeat (ITR), anucleotide sequence of interest (for example an expression cassette asdescribed herein) and a second AAV ITR, where the first ITR (5′ ITR) andthe second ITR (3′ ITR) are symmetric, or substantially symmetrical withrespect to each other—that is, a ceDNA vector can comprise ITR sequencesthat have a symmetrical three-dimensional spatial organization such thattheir structure is the same shape in geometrical space, or have the sameA, C-C′ and B-B′ loops in 3D space. In such an embodiment, a symmetricalITR pair, or substantially symmetrical ITR pair can be modified ITRs(e.g., mod-ITRs) that are not wild-type ITRs. A mod-ITR pair can havethe same sequence which has one or more modifications from wild-type ITRand are reverse complements (inverted) of each other. In alternativeembodiments, a modified ITR pair are substantially symmetrical asdefined herein, that is, the modified ITR pair can have a differentsequence but have corresponding or the same symmetricalthree-dimensional shape.

(i) Wildtype ITRs

In some embodiments, the symmetrical ITRs, or substantially symmetricalITRs are wild type (WT-ITRs) as described herein. That is, both ITRshave a wild type sequence, but do not necessarily have to be WT-ITRsfrom the same AAV serotype. That is, in some embodiments, one WT-ITR canbe from one AAV serotype, and the other WT-ITR can be from a differentAAV serotype. In such an embodiment, a WT-ITR pair are substantiallysymmetrical as defined herein, that is, they can have one or moreconservative nucleotide modification while still retaining thesymmetrical three-dimensional spatial organization.

Accordingly, as disclosed herein, ceDNA vectors contain a transgene orheterologous nucleic acid sequence positioned between two flankingwild-type inverted terminal repeat (WT-ITR) sequences, that are eitherthe reverse complement (inverted) of each other, or alternatively, aresubstantially symmetrical relative to each other—that is a WT-ITR pairhave symmetrical three-dimensional spatial organization. In someembodiments, a wild-type ITR sequence (e.g. AAV WT-ITR) comprises afunctional Rep binding site (RBS; e.g. 5′-GCGCGCTCGCTCGCTC-3′ for AAV2,SEQ ID NO: 60) and a functional terminal resolution site (TRS; e.g.5′-AGTT-3′, SEQ ID NO: 62).

In one aspect, ceDNA vectors for expression of FVIII protein areobtainable from a vector polynucleotide that encodes a heterologousnucleic acid operatively positioned between two WT inverted terminalrepeat sequences (WT-ITRs) (e.g. AAV WT-ITRs). That is, both ITRs have awild type sequence, but do not necessarily have to be WT-ITRs from thesame AAV serotype. That is, in some embodiments, one WT-ITR can be fromone AAV serotype, and the other WT-ITR can be from a different AAVserotype. In such an embodiment, the WT-ITR pair are substantiallysymmetrical as defined herein, that is, they can have one or moreconservative nucleotide modification while still retaining thesymmetrical three-dimensional spatial organization. In some embodiments,the 5′ WT-ITR is from one AAV serotype, and the 3′ WT-ITR is from thesame or a different AAV serotype. In some embodiments, the 5′ WT-ITR andthe 3′WT-ITR are mirror images of each other, that is they aresymmetrical. In some embodiments, the 5′ WT-ITR and the 3′ WT-ITR arefrom the same AAV serotype.

WT ITRs are well known. In one embodiment the two ITRs are from the sameAAV2 serotype. In certain embodiments one can use WT from otherserotypes. There are a number of serotypes that are homologous, e.g.AAV2, AAV4, AAV6, AAV8. In one embodiment, closely homologous ITRs (e.g.ITRs with a similar loop structure) can be used. In another embodiment,one can use AAV WT ITRs that are more diverse, e.g., AAV2 and AAV5, andstill another embodiment, one can use an ITR that is substantiallyWT—that is, it has the basic loop structure of the WT but someconservative nucleotide changes that do not alter or affect theproperties. When using WT-ITRs from the same viral serotype, one or moreregulatory sequences may further be used. In certain embodiments, theregulatory sequence is a regulatory switch that permits modulation ofthe activity of the ceDNA, e.g., the expression of the encoded FVIIIprotein.

In some embodiments, one aspect of the technology described hereinrelates to a ceDNA vector for expression of FVIII protein, wherein theceDNA vector comprises at least one heterologous nucleotide sequenceencoding the FVIII protein, operably positioned between two wild-typeinverted terminal repeat sequences (WT-ITRs), wherein the WT-ITRs can befrom the same serotype, different serotypes or substantially symmetricalwith respect to each other (i.e., have the symmetrical three-dimensionalspatial organization such that their structure is the same shape ingeometrical space, or have the same A, C-C′ and B-B′ loops in 3D space).In some embodiments, the symmetric WT-ITRs comprises a functionalterminal resolution site and a Rep binding site. In some embodiments,the heterologous nucleic acid sequence encodes a transgene, and whereinthe vector is not in a viral capsid.

In some embodiments, the WT-ITRs are the same but the reverse complementof each other. For example, the sequence AACG in the 5′ ITR may be CGTT(i.e., the reverse complement) in the 3′ ITR at the corresponding site.In one example, the 5′ WT-ITR sense strand comprises the sequence ofATCGATCG and the corresponding 3′ WT-ITR sense strand comprises CGATCGAT(i.e., the reverse complement of ATCGATCG). In some embodiments, theWT-ITRs ceDNA further comprises a terminal resolution site and areplication protein binding site (RPS) (sometimes referred to as areplicative protein binding site), e.g. a Rep binding site.

Exemplary WT-ITR sequences for use in the ceDNA vectors for expressionof FVIII protein comprising WT-ITRs are shown in Table 3 herein, whichshows pairs of WT-ITRs (5′ WT-ITR and the 3′ WT-ITR).

As an exemplary example, the present disclosure provides a ceDNA vectorfor expression of FVIII protein comprising a promoter operably linked toa transgene (e.g., heterologous nucleic acid sequence), with or withoutthe regulatory switch, where the ceDNA is devoid of capsid proteins andis: (a) produced from a ceDNA-plasmid (e.g., see FIGS. 1F-1G) thatencodes WT-ITRs, where each WT-ITR has the same number ofintramolecularly duplexed base pairs in its hairpin secondaryconfiguration (preferably excluding deletion of any AAA or TTT terminalloop in this configuration compared to these reference sequences), and(b) is identified as ceDNA using the assay for the identification ofceDNA by agarose gel electrophoresis under native gel and denaturingconditions in Example 1.

In some embodiments, the flanking WT-ITRs are substantially symmetricalto each other. In this embodiment the 5′ WT-ITR can be from one serotypeof AAV, and the 3′ WT-ITR from a different serotype of AAV, such thatthe WT-ITRs are not identical reverse complements. For example, the 5′WT-ITR can be from AAV2, and the 3′ WT-ITR from a different serotype(e.g. AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments,WT-ITRs can be selected from two different parvoviruses selected fromany to of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus),bovine parvovirus, goat parvovirus, avian parvovirus, canine parvovirus,equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV.In some embodiments, such a combination of WT ITRs is the combination ofWT-ITRs from AAV2 and AAV6. In one embodiment, the substantiallysymmetrical WT-ITRs are when one is inverted relative to the other ITRat least 90% identical, at least 95% identical, at least 96% . . . 97% .. . 98% . . . 99% . . . 99.5% and all points in between, and has thesame symmetrical three-dimensional spatial organization. In someembodiments, a WT-ITR pair are substantially symmetrical as they havesymmetrical three-dimensional spatial organization, e.g., have the same3D organization of the A, C-C′, B-B′ and D arms. In one embodiment, asubstantially symmetrical WT-ITR pair are inverted relative to theother, and are at least 95% identical, at least 96% . . . 97% . . . 98%. . . 99% . . . 99.5% and all points in between, to each other, and oneWT-ITR retains the Rep-binding site (RBS) of 5′-GCGCGCTCGCTCGCTC-3′ (SEQID NO: 60) and a terminal resolution site (TRS). In some embodiments, asubstantially symmetrical WT-ITR pair are inverted relative to eachother, and are at least 95% identical, at least 96% . . . 97% . . . 98%. . . 99% . . . 99.5% and all points in between, to each other, and oneWT-ITR retains the Rep-binding site (RBS) of 5′-GCGCGCTCGCTCGCTC-3′ (SEQID NO: 60) and a terminal resolution site (TRS) and in addition to avariable palindromic sequence allowing for hairpin secondary structureformation. Homology can be determined by standard means well known inthe art such as BLAST (Basic Local Alignment Search Tool), BLASTN atdefault setting.

In some embodiments, the structural element of the ITR can be anystructural element that is involved in the functional interaction of theITR with a large Rep protein (e.g., Rep 78 or Rep 68). In certainembodiments, the structural element provides selectivity to theinteraction of an ITR with a large Rep protein, i.e., determines atleast in part which Rep protein functionally interacts with the ITR. Inother embodiments, the structural element physically interacts with alarge Rep protein when the Rep protein is bound to the ITR. Eachstructural element can be, e.g., a secondary structure of the ITR, anucleotide sequence of the ITR, a spacing between two or more elements,or a combination of any of the above. In one embodiment, the structuralelements are selected from the group consisting of an A and an A′ arm, aB and a B′ arm, a C and a C′ arm, a D arm, a Rep binding site (RBE) andan RBE′ (i.e., complementary RBE sequence), and a terminal resolutionsire (TRS).

By way of example only, Table 2 indicates exemplary combinations ofWT-ITRs.

Table 2: Exemplary combinations of WT-ITRs from the same serotype ordifferent serotypes, or different parvoviruses. The order shown is notindicative of the ITR position, for example, “AAV1, AAV2” demonstratesthat the ceDNA can comprise a WT-AAV1 ITR in the 5′ position, and aWT-AAV2 ITR in the 3′ position, or vice versa, a WT-AAV2 ITR the 5′position, and a WT-AAV1 ITR in the 3′ position. Abbreviations: AAVserotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAVserotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAVserotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAVserotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12(AAV12); AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome (E.g., NCBI: NC002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261),ITRs from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV),canine, equine, and ovine AAV), ITRs from B19 parvoviris (GenBankAccession No: NC 000883), Minute Virus from Mouse (MVM) (GenBankAccession No. NC 001510); Goose: goose parvovirus (GenBank Accession No.NC 001701); snake: snake parvovirus 1 (GenBank Accession No. NC 006148).

TABLE 2 AAV1, AAV1 AAV2, AAV2 AAV3, AAV3 AAV4, AAV4 AAV5, AAV5 AAV1,AAV2 AAV2, AAV3 AAV3, AAV4 AAV4, AAV5 AAV5, AAV6 AAV1, AAV3 AAV2, AAV4AAV3, AAV5 AAV4, AAV6 AAV5, AAV7 AAV1, AAV4 AAV2, AAV5 AAV3, AAV6 AAV4,AAV7 AAV5, AAV8 AAV1, AAV5 AAV2, AAV6 AAV3, AAV7 AAV4, AAV8 AAV5, AAV9AAV1, AAV6 AAV2, AAV7 AAV3, AAV8 AAV4, AAV9 AAV5, AAV10 AAV1, AAV7 AAV2,AAV8 AAV3, AAV9 AAV4, AAV10 AAV5, AAV11 AAV1, AAV8 AAV2, AAV9 AAV3,AAV10 AAV4, AAV11 AAV5, AAV12 AAV1, AAV9 AAV2, AAV10 AAV3, AAV11 AAV4,AAV12 AAV5, AAVRH8 AAV1, AAV10 AAV2, AAV11 AAV3, AAV12 AAV4, AAVRH8AAV5, AAVRH10 AAV1, AAV11 AAV2, AAV12 AAV3, AAVRH8 AAV4, AAVRH10 AAV5,AAV13 AAV1, AAV12 AAV2, AAVRH8 AAV3, AAVRH10 AAV4, AAV13 AAV5, AAVDJAAV1, AAVRH8 AAV2, AAVRH10 AAV3, AAV13 AAV4, AAVDJ AAV5, AAVDJ8 AAV1,AAVRH10 AAV2, AAV13 AAV3, AAVDJ AAV4, AAVDJ8 AAV5, AVIAN AAV1, AAV13AAV2, AAVDJ AAV3, AAVDJ8 AAV4, AVIAN AAV5, BOVINE AAV1, AAVDJ AAV2,AAVDJ8 AAV3, AVIAN AAV4, BOVINE AAV5, CANINE AAV1, AAVDJ8 AAV2, AVIANAAV3, BOVINE AAV4, CANINE AAV5, EQUINE AAV1, AVIAN AAV2, BOVINE AAV3,CANINE AAV4, EQUINE AAV5, GOAT AAV1, BOVINE AAV2, CANINE AAV3, EQUINEAAV4, GOAT AAV5, SHRIMP AAV1, CANINE AAV2, EQUINE AAV3, GOAT AAV4,SHRIMP AAV5, PORCINE AAV1, EQUINE AAV2, GOAT AAV3, SHRIMP AAV4, PORCINEAAV5, INSECT AAV1, GOAT AAV2, SHRIMP AAV3, PORCINE AAV4, INSECT AAV5,OVINE AAV1, SHRIMP AAV2, PORCINE AAV3, INSECT AAV4, OVINE AAV5, B19AAV1, PORCINE AAV2, INSECT AAV3, OVINE AAV4, B19 AAV5, MVM AAV1, INSECTAAV2, OVINE AAV3, B19 AAV4, MVM AAV5, GOOSE AAV1, OVINE AAV2, B19 AAV3,MVM AAV4, GOOSE AAV5, SNAKE AAV1, B19 AAV2, MVM AAV3, GOOSE AAV4, SNAKEAAV1, MVM AAV2, GOOSE AAV3, SNAKE AAV1, GOOSE AAV2, SNAKE AAV1, SNAKEAAV6, AAV6 AAV7, AAV7 AAV8, AAV8 AAV9, AAV9 AAV10, AAV10 AAV6, AAV7AAV7, AAV8 AAV8, AAV9 AAV9, AAV10 AAV10, AAV11 AAV6, AAV8 AAV7, AAV9AAV8, AAV10 AAV9, AAV11 AAV10, AAV12 AAV6, AAV9 AAV7, AAV10 AAV8, AAV11AAV9, AAV12 AAV10, AAVRH8 AAV6, AAV10 AAV7, AAV11 AAV8, AAV12 AAV9,AAVRH8 AAV10, AAVRH10 AAV6, AAV11 AAV7, AAV12 AAV8, AAVRH8 AAV9, AAVRH10AAV10, AAV13 AAV6, AAV12 AAV7, AAVRH8 AAV8, AAVRH10 AAV9, AAV13 AAV10,AAVDJ AAV6, AAVRH8 AAV7, AAVRH10 AAV8, AAV13 AAV9, AAVDJ AAV10, AAVDJ8AAV6, AAVRH10 AAV7, AAV13 AAV8, AAVDJ AAV9, AAVDJ8 AAV10, AVIAN AAV6,AAV13 AAV7, AAVDJ AAV8, AAVDJ8 AAV9, AVIAN AAV10, BOVINE AAV6, AAVDJAAV7, AAVDJ8 AAV8, AVIAN AAV9, BOVINE AAV10, CANINE AAV6, AAVDJ8 AAV7,AVIAN AAV8, BOVINE AAV9, CANINE AAV10, EQUINE AAV6, AVIAN AAV7, BOVINEAAV8, CANINE AAV9, EQUINE AAV10, GOAT AAV6, BOVINE AAV7, CANINE AAV8,EQUINE AAV9, GOAT AAV10, SHRIMP AAV6, CANINE AAV7, EQUINE AAV8, GOATAAV9, SHRIMP AAV10, PORCINE AAV6, EQUINE AAV7, GOAT AAV8, SHRIMP AAV9,PORCINE AAV10, INSECT AAV6, GOAT AAV7, SHRIMP AAV8, PORCINE AAV9, INSECTAAV10, OVINE AAV6, SHRIMP AAV7, PORCINE AAV8, INSECT AAV9, OVINE AAV10,B19 AAV6, PORCINE AAV7, INSECT AAV8, OVINE AAV9, B19 AAV10, MVM AAV6,INSECT AAV7, OVINE AAV8, B19 AAV9, MVM AAV10, GOOSE AAV6, OVINE AAV7,B19 AAV8, MVM AAV9, GOOSE AAV10, SNAKE AAV6, B19 AAV7, MVM AAV8, GOOSEAAV9, SNAKE AAV6, MVM AAV7, GOOSE AAV8, SNAKE AAV6, GOOSE AAV7, SNAKEAAV6, SNAKE AAV11, AAV11 AAV12, AAV12 AAVRH8, AAVRH8 AAVRH10, AAVRH10AAV13, AAV13 AAV11, AAV12 AAV12, AAVRH8 AAVRH8, AAVRH10 AAVRH10, AAV13AAV13, AAVDJ AAV11, AAVRH8 AAV12, AAVRH10 AAVRH8, AAV13 AAVRH10, AAVDJAAV13, AAVDJ8 AAV11, AAVRH10 AAV12, AAV13 AAVRH8, AAVDJ AAVRH10, AAVDJ8AAV13, AVIAN AAV11, AAV13 AAV12, AAVDJ AAVRH8, AAVDJ8 AAVRH10, AVIANAAV13, BOVINE AAV11, AAVDJ AAV12, AAVDJ8 AAVRH8, AVIAN AAVRH10, BOVINEAAV13, CANINE AAV11, AAVDJ8 AAV12, AVIAN AAVRH8, BOVINE AAVRH10, CANINEAAV13, EQUINE AAV11, AVIAN AAV12, BOVINE AAVRH8, CANINE AAVRH10, EQUINEAAV13, GOAT AAV11, BOVINE AAV12, CANINE AAVRH8, EQUINE AAVRH10, GOATAAV13, SHRIMP AAV11, CANINE AAV12, EQUINE AAVRH8, GOAT AAVRH10, SHRIMPAAV13, PORCINE AAV11, EQUINE AAV12, GOAT AAVRH8, SHRIMP AAVRH10, PORCINEAAV13, INSECT AAV11, GOAT AAV12, SHRIMP AAVRH8, PORCINE AAVRH10, INSECTAAV13, OVINE AAV11, SHRIMP AAV12, PORCINE AAVRH8, INSECT AAVRH10, OVINEAAV13, B19 AAV11, PORCINE AAV12, INSECT AAVRH8, OVINE AAVRH10, B19AAV13, MVM AAV11, INSECT AAV12, OVINE AAVRH8, B19 AAVRH10, MVM AAV13,GOOSE AAV11, OVINE AAV12, B19 AAVRH8, MVM AAVRH10, GOOSE AAV13, SNAKEAAV11, B19 AAV12, MVM AAVRH8, GOOSE AAVRH10, SNAKE AAV11, MVM AAV12,GOOSE AAVRH8, SNAKE AAV11, GOOSE AAV12, SNAKE AAV11, SNAKE AAVDJ, AAVDJAAVDJ8, AVVDJ8 AVIAN, AVIAN BOVINE, BOVINE CANINE, CANINE AAVDJ, AAVDJ8AAVDJ8, AVIAN AVIAN, BOVINE BOVINE, CANINE CANINE, EQUINE AAVDJ, AVIANAAVDJ8, BOVINE AVIAN, CANINE BOVINE, EQUINE CANINE, GOAT AAVDJ, BOVINEAAVDJ8, CANINE AVIAN, EQUINE BOVINE, GOAT CANINE, SHRIMP AAVDJ, CANINEAAVDJ8, EQUINE AVIAN, GOAT BOVINE, SHRIMP CANINE, PORCINE AAVDJ, EQUINEAAVDJ8, GOAT AVIAN, SHRIMP BOVINE, PORCINE CANINE, INSECT AAVDJ, GOATAAVDJ8, SHRIMP AVIAN, PORCINE BOVINE, INSECT CANINE, OVINE AAVDJ, SHRIMPAAVDJ8, PORCINE AVIAN, INSECT BOVINE, OVINE CANINE, B19 AAVDJ, PORCINEAAVDJ8, INSECT AVIAN, OVINE BOVINE, B19 CANINE, MVM AAVDJ, INSECTAAVDJ8, OVINE AVIAN, B19 BOVINE, MVM CANINE, GOOSE AAVDJ, OVINE AAVDJ8,B19 AVIAN, MVM BOVINE, GOOSE CANINE, SNAKE AAVDJ, B19 AAVDJ8, MVM AVIAN,GOOSE BOVINE, SNAKE AAVDJ, MVM AAVDJ8, GOOSE AVIAN, SNAKE AAVDJ, GOOSEAAVDJ8, SNAKE AAVDJ, SNAKE EQUINE, EQUINE GOAT, GOAT SHRIMP, SHRIMPPORCINE, PORCINE INSECT, INSECT EQUINE, GOAT GOAT, SHRIMP SHRIMP,PORCINE PORCINE, INSECT INSECT, OVINE EQUINE, SHRIMP GOAT, PORCINESHRIMP, INSECT PORCINE, OVINE INSECT, B19 EQUINE, PORCINE GOAT, INSECTSHRIMP, OVINE PORCINE, B19 INSECT, MVM EQUINE, INSECT GOAT, OVINESHRIMP, B19 PORCINE, MVM INSECT, GOOSE EQUINE, OVINE GOAT, B19 SHRIMP,MVM PORCINE, GOOSE INSECT, SNAKE EQUINE, B19 GOAT, MVM SHRIMP, GOOSEPORCINE, SNAKE EQUINE, MVM GOAT, GOOSE SHRIMP, SNAKE EQUINE, GOOSE GOAT,SNAKE EQUINE, SNAKE OVINE, OVINE B19, B19 MVM, MVM GOOSE, GOOSE SNAKE,SNAKE OVINE, B19 B19, MVM MVM, GOOSE GOOSE, SNAKE OVINE, MVM B19, GOOSEMVM, SNAKE OVINE, GOOSE B19, SNAKE OVINE, SNAKE

By way of example only, Table 3 shows the sequences of exemplary WT-ITRsfrom some different AAV serotypes.

TABLE 3 AAV serotype 5′ WT-ITR (LEFT) 3′ WT-ITR (RIGHT) AAV1 5′- 5′-TTGCCCACTCCCTCTCTGCGCGCTCGC TTACCCTAGTGATGGAGTTGCCCACTCTCGCTCGGTGGGGCCTGCGGACCAAA CCTCTCTGCGCGCGTCGCTCGCTCGGTGGTCCGCAGACGGCAGAGGTCTCCTCT GGGGCCGGCAGAGGAGACCTCTGCCGGCCGGCCCCACCGAGCGAGCGACGCG TCTGCGGACCTTTGGTCCGCAGGCCCCCGCAGAGAGGGAGTGGGCAACTCCAT ACCGAGCGAGCGAGCGCGCAGAGAGG CACTAGGGTAA-3′GAGTGGGCAA-3′ (SEQ ID NO: 10) (SEQ ID NO: 5) AAV2CCTGCAGGCAGCTGCGCGCTCGCTCGC AGGAACCCCTAGTGATGGAGTTGGCCATCACTGAGGCCGCCCGGGCAAAGCCC CTCCCTCTCTGCGCGCTCGCTCGCTCACGGGCGTCGGGCGACCTTTGGTCGCCCG TGAGGCCGGGCGACCAAAGGTCGCCCGCCTCAGTGAGCGAGCGAGCGCGCAG GACGCCCGGGCTTTGCCCGGGCGGCCTAGAGGGAGTGGCCAACTCCATCACTA CAGTGAGCGAGCGAGCGCGCAGCTGCGGGGTTCCT (SEQ ID NO: 2) CTGCAGG (SEQ ID NO: 1) AAV3 5′- 5′-TTGGCCACTCCCTCTATGCGCACTCGC ATACCTCTAGTGATGGAGTTGGCCACTTCGCTCGGTGGGGCCTGGCGACCAAA CCCTCTATGCGCACTCGCTCGCTCGGTGGTCGCCAGACGGACGTGGGTTTCCAC GGGGCCGGACGTGGAAACCCACGTCCGTCCGGCCCCACCGAGCGAGCGAGTG GTCTGGCGACCTTTGGTCGCCAGGCCCCGCATAGAGGGAGTGGCCAACTCCAT CACCGAGCGAGCGAGTGCGCATAGAGCACTAGAGGTAT-3′ (SEQ ID NO: 6) GGAGTGGCCAA-3′ (SEQ ID NO: 11) AAV4 5′-5′- TTGGCCACTCCCTCTATGCGCGCTCGC AGTTGGCCACATTAGCTATGCGCGCTCTCACTCACTCGGCCCTGGAGACCAAAG GCTCACTCACTCGGCCCTGGAGACCAAGTCTCCAGACTGCCGGCCTCTGGCCGG AGGTCTCCAGACTGCCGGCCTCTGGCCCAGGGCCGAGTGAGTGAGCGAGCGCG GGCAGGGCCGAGTGAGTGAGCGAGCGCATAGAGGGAGTGGCCAACT-3′ (SEQ CGCATAGAGGGAGTGGCCAA-3′ (SEQ ID NO: 7)ID NO: 12) AAV5 5′- 5′- TCCCCCCTGTCGCGTTCGCTCGCTCGCCTTACAAAACCCCCTTGCTTGAGAGTG TGGCTCGTTTGGGGGGGCGACGGCCATGGCACTCTCCCCCCTGTCGCGTTCGCT GAGGGCCGTCGTCTGGCAGCTCTTTGACGCTCGCTGGCTCGTTTGGGGGGGTGG GCTGCCACCCCCCCAAACGAGCCAGCCAGCTCAAAGAGCTGCCAGACGACGG GAGCGAGCGAACGCGACAGGGGGGAGCCCTCTGGCCGTCGCCCCCCCAAACGA AGTGCCACACTCTCAAGCAAGGGGGTGCCAGCGAGCGAGCGAACGCGACAGG TTTGTAAG-3′ (SEQ ID NO: 8)GGGGA-3′ (SEQ ID NO: 13) AAV6 5′- 5′- TTGCCCACTCCCTCTAATGCGCGCTCGATACCCCTAGTGATGGAGTTGCCCACT CTCGCTCGGTGGGGCCTGCGGACCAACCCTCTATGCGCGCTCGCTCGCTCGGT AGGTCCGCAGACGGCAGAGGTCTCCTGGGGCCGGCAGAGGAGACCTCTGCCG CTGCCGGCCCCACCGAGCGAGCGAGCTCTGCGGACCTTTGGTCCGCAGGCCCC GCGCATAGAGGGAGTGGGCAACTCCAACCGAGCGAGCGAGCGCGCATTAGAG TCACTAGGGGTAT-3′ (SEQ ID NO: 9)GGAGTGGGCAA (SEQ ID NO: 14)

In some embodiments, the nucleotide sequence of the WT-ITR sequence canbe modified (e.g., by modifying 1, 2, 3, 4 or 5, or more nucleotides orany range therein), whereby the modification is a substitution for acomplementary nucleotide, e.g., G for a C, and vice versa, and T for anA, and vice versa.

In certain embodiments of the present invention, the ceDNA vector forexpression of FVIII protein does not have a WT-ITR consisting of thenucleotide sequence selected from any of: SEQ ID NOs: 1, 2, 5-14. Inalternative embodiments of the present invention, if a ceDNA vector hasa WT-ITR comprising the nucleotide sequence selected from any of: SEQ IDNOs: 1, 2, 5-14, then the flanking ITR is also WT and the ceDNA vectorcomprises a regulatory switch, e.g., as disclosed herein and inInternational application PCT/US18/49996 (e.g., see Table 11 ofPCT/US18/49996). In some embodiments, the ceDNA vector for expression ofFVIII protein comprises a regulatory switch as disclosed herein and aWT-ITR selected having the nucleotide sequence selected from any of thegroup consisting of: SEQ ID NO: 1, 2, 5-14.

The ceDNA vector for expression of FVIII protein as described herein caninclude WT-ITR structures that retains an operable RBE, TRS and RBE′portion. FIG. 2A and FIG. 2B, using wild-type ITRs for exemplarypurposes, show one possible mechanism for the operation of a TRS sitewithin a wild type ITR structure portion of a ceDNA vector. In someembodiments, the ceDNA vector for expression of FVIII protein containsone or more functional WT-ITR polynucleotide sequences that comprise aRep-binding site (RBS; 5′-GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60) for AAV2)and a terminal resolution site (TRS; 5′-AGTT (SEQ ID NO: 62)). In someembodiments, at least one WT-ITR is functional. In alternativeembodiments, where a ceDNA vector for expression of FVIII proteincomprises two WT-ITRs that are substantially symmetrical to each other,at least one WT-ITR is functional and at least one WT-ITR isnon-functional.

B. Modified ITRs (Mod-ITRs) in General for ceDNA Vectors ComprisingAsymmetric ITR Pairs or Symmetric ITR Pairs

As discussed herein, a ceDNA vector for expression of FVIII protein cancomprise a symmetrical ITR pair or an asymmetrical ITR pair. In bothinstances, one or both of the ITRs can be modified ITRs—the differencebeing that in the first instance (i.e., symmetric mod-ITRs), themod-ITRs have the same three-dimensional spatial organization (i.e.,have the same A-A′, C-C′ and B-B′ arm configurations), whereas in thesecond instance (i.e., asymmetric mod-ITRs), the mod-ITRs have adifferent three-dimensional spatial organization (i.e., have a differentconfiguration of A-A′, C-C′ and B-B′ arms).

In some embodiments, a modified ITR is an ITRs that is modified bydeletion, insertion, and/or substitution as compared to a wild-type ITRsequence (e.g. AAV ITR). In some embodiments, at least one of the ITRsin the ceDNA vector comprises a functional Rep binding site (RBS; e.g.5′-GCGCGCTCGCTCGCTC-3′ for AAV2, SEQ ID NO: 60) and a functionalterminal resolution site (TRS; e.g. 5′-AGTT-3′, SEQ ID NO: 62.) In oneembodiment, at least one of the ITRs is a non-functional ITR. In oneembodiment, the different or modified ITRs are not each wild type ITRsfrom different serotypes.

Specific alterations and mutations in the ITRs are described in detailherein, but in the context of ITRs, “altered” or “mutated” or“modified”, it indicates that nucleotides have been inserted, deleted,and/or substituted relative to the wild-type, reference, or original ITRsequence. The altered or mutated ITR can be an engineered ITR. As usedherein, “engineered” refers to the aspect of having been manipulated bythe hand of man. For example, a polypeptide is considered to be“engineered” when at least one aspect of the polypeptide, e.g., itssequence, has been manipulated by the hand of man to differ from theaspect as it exists in nature.

In some embodiments, a mod-ITR may be synthetic. In one embodiment, asynthetic ITR is based on ITR sequences from more than one AAV serotype.In another embodiment, a synthetic ITR includes no AAV-based sequence.In yet another embodiment, a synthetic ITR preserves the ITR structuredescribed above although having only some or no AAV-sourced sequence. Insome aspects, a synthetic ITR may interact preferentially with a wildtype Rep or a Rep of a specific serotype, or in some instances will notbe recognized by a wild-type Rep and be recognized only by a mutatedRep.

The skilled artisan can determine the corresponding sequence in otherserotypes by known means. For example, determining if the change is inthe A, A′, B, B′, C, C′ or D region and determine the correspondingregion in another serotype. One can use BLAST® (Basic Local AlignmentSearch Tool) or other homology alignment programs at default status todetermine the corresponding sequence. The invention further providespopulations and pluralities of ceDNA vectors comprising mod-ITRs from acombination of different AAV serotypes—that is, one mod-ITR can be fromone AAV serotype and the other mod-ITR can be from a different serotype.Without wishing to be bound by theory, in one embodiment one ITR can befrom or based on an AAV2 ITR sequence and the other ITR of the ceDNAvector can be from or be based on any one or more ITR sequence of AAVserotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAVserotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAVserotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), orAAV serotype 12 (AAV12).

Any parvovirus ITR can be used as an ITR or as a base ITR formodification. Preferably, the parvovirus is a dependovirus. Morepreferably AAV. The serotype chosen can be based upon the tissue tropismof the serotype. AAV2 has a broad tissue tropism, AAV1 preferentiallytargets to neuronal and skeletal muscle, and AAV5 preferentially targetsneuronal, retinal pigmented epithelia, and photoreceptors. AAV6preferentially targets skeletal muscle and lung. AAV8 preferentiallytargets liver, skeletal muscle, heart, and pancreatic tissues. AAV9preferentially targets liver, skeletal and lung tissue. In oneembodiment, the modified ITR is based on an AAV2 ITR.

More specifically, the ability of a structural element to functionallyinteract with a particular large Rep protein can be altered by modifyingthe structural element. For example, the nucleotide sequence of thestructural element can be modified as compared to the wild-type sequenceof the ITR. In one embodiment, the structural element (e.g., A arm, A′arm, B arm, B′ arm, C arm, C′ arm, D arm, RBE, RBE′, and TRS) of an ITRcan be removed and replaced with a wild-type structural element from adifferent parvovirus. For example, the replacement structure can be fromAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovineparvovirus, goat parvovirus, avian parvovirus, canine parvovirus, equineparvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. Forexample, the ITR can be an AAV2 ITR and the A or A′ arm or RBE can bereplaced with a structural element from AAV5. In another example, theITR can be an AAV5 ITR and the C or C′ arms, the RBE, and the TRS can bereplaced with a structural element from AAV2. In another example, theAAV ITR can be an AAV5 ITR with the B and B′ arms replaced with the AAV2ITR B and B′ arms.

By way of example only, Table 4 indicates exemplary modifications of atleast one nucleotide (e.g., a deletion, insertion and/or substitution)in regions of a modified ITR, where X is indicative of a modification ofat least one nucleic acid (e.g., a deletion, insertion and/orsubstitution) in that section relative to the corresponding wild-typeITR. In some embodiments, any modification of at least one nucleotide(e.g., a deletion, insertion and/or substitution) in any of the regionsof C and/or C′ and/or B and/or B′ retains three sequential T nucleotides(i.e., TTT) in at least one terminal loop. For example, if themodification results in any of: a single arm ITR (e.g., single C-C′ arm,or a single B-B′ arm), or a modified C-B′ arm or C′-B arm, or a two armITR with at least one truncated arm (e.g., a truncated C-C′ arm and/ortruncated B-B′ arm), at least the single arm, or at least one of thearms of a two arm ITR (where one arm can be truncated) retains threesequential T nucleotides (i.e., TTT) in at least one terminal loop. Insome embodiments, a truncated C-C′ arm and/or a truncated B-B′ arm hasthree sequential T nucleotides (i.e., TTT) in the terminal loop.

TABLE 4 Exemplary combinations of modifications of at least onenucleotide (e.g., a deletion, insertion and/or substitution) todifferent B-B’ and C-C’ regions or arms of ITRs (X indicates anucleotide modification, e.g., addition, deletion or substitution of atleast one nucleotide in the region). B region B’ region C region C’region X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

In some embodiments, mod-ITR for use in a ceDNA vector for expression ofFVIII protein comprises an asymmetric ITR pair, or a symmetric mod-ITRpair as disclosed herein, can comprise any one of the combinations ofmodifications shown in Table 4, and also a modification of at least onenucleotide in any one or more of the regions selected from: between A′and C, between C and C′, between C′ and B, between B and B′ and betweenB′ and A. In some embodiments, any modification of at least onenucleotide (e.g., a deletion, insertion and/or substitution) in the C orC′ or B or B′ regions, still preserves the terminal loop of thestem-loop. In some embodiments, any modification of at least onenucleotide (e.g., a deletion, insertion and/or substitution) between Cand C′ and/or B and B′ retains three sequential T nucleotides (i.e.,TTT) in at least one terminal loop. In alternative embodiments, anymodification of at least one nucleotide (e.g., a deletion, insertionand/or substitution) between C and C′ and/or B and B′ retains threesequential A nucleotides (i.e., AAA) in at least one terminal loop. Insome embodiments, a modified ITR for use herein can comprise any one ofthe combinations of modifications shown in Table 4, and also amodification of at least one nucleotide (e.g., a deletion, insertionand/or substitution) in any one or more of the regions selected from:A′, A and/or D. For example, in some embodiments, a modified ITR for useherein can comprise any one of the combinations of modifications shownin Table 4, and also a modification of at least one nucleotide (e.g., adeletion, insertion and/or substitution) in the A region. In someembodiments, a modified ITR for use herein can comprise any one of thecombinations of modifications shown in Table 4, and also a modificationof at least one nucleotide (e.g., a deletion, insertion and/orsubstitution) in the A′ region. In some embodiments, a modified ITR foruse herein can comprise any one of the combinations of modificationsshown in Table 4, and also a modification of at least one nucleotide(e.g., a deletion, insertion and/or substitution) in the A and/or A′region. In some embodiments, a modified ITR for use herein can compriseany one of the combinations of modifications shown in Table 4, and alsoa modification of at least one nucleotide (e.g., a deletion, insertionand/or substitution) in the D region.

In one embodiment, the nucleotide sequence of the structural element canbe modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides or any rangetherein) to produce a modified structural element. In one embodiment,the specific modifications to the ITRs are exemplified herein (e.g., SEQID NOS: 3, 4, 15-47, 101-116 or 165-187, or shown in FIG. 7A-7B ofPCT/US2018/064242, filed on Dec. 6, 2018 (e.g., SEQ ID Nos 97-98,101-103, 105-108, 111-112, 117-134, 545-54 in PCT/US2018/064242). Insome embodiments, an ITR can be modified (e.g., by modifying 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or morenucleotides or any range therein). In other embodiments, the ITR canhave at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or more sequence identitywith one of the modified ITRs of SEQ ID NOS: 3, 4, 15-47, 101-116 or165-187, or the RBE-containing section of the A-A′ arm and C-C′ and B-B′arms of SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187, or shown in Tables2-9 (i.e., SEQ ID NO: 110-112, 115-190, 200-468) of Internationalapplication PCT/US18/49996, which is incorporated herein in its entiretyby reference.

In some embodiments, a modified ITR can for example, comprise removal ordeletion of all of a particular arm, e.g., all or part of the A-A′ arm,or all or part of the B-B′ arm or all or part of the C-C′ arm, oralternatively, the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more basepairs forming the stem of the loop so long as the final loop capping thestem (e.g., single arm) is still present (e.g., see ITR-21 in FIG. 7A ofPCT/US2018/064242, filed Dec. 6, 2018). In some embodiments, a modifiedITR can comprise the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more basepairs from the B-B′ arm. In some embodiments, a modified ITR cancomprise the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairsfrom the C-C′ arm (see, e.g., ITR-1 in FIG. 3B, or ITR-45 in FIG. 7A ofPCT/US2018/064242, filed Dec. 6, 2018). In some embodiments, a modifiedITR can comprise the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more basepairs from the C-C′ arm and the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 ormore base pairs from the B-B′ arm. Any combination of removal of basepairs is envisioned, for example, 6 base pairs can be removed in theC-C′ arm and 2 base pairs in the B-B′ arm. As an illustrative example,FIG. 3B shows an exemplary modified ITR with at least 7 base pairsdeleted from each of the C portion and the C′ portion, a substitution ofa nucleotide in the loop between C and C′ region, and at least one basepair deletion from each of the B region and B′ regions such that themodified ITR comprises two arms where at least one arm (e.g., C-C′) istruncated. In some embodiments, the modified ITR also comprises at leastone base pair deletion from each of the B region and B′ regions, suchthat the B-B′ arm is also truncated relative to WT ITR.

In some embodiments, a modified ITR can have between 1 and 50 (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletionsrelative to a full-length wild-type ITR sequence. In some embodiments, amodified ITR can have between 1 and 30 nucleotide deletions relative toa full-length WT ITR sequence. In some embodiments, a modified ITR hasbetween 2 and 20 nucleotide deletions relative to a full-lengthwild-type ITR sequence.

In some embodiments, a modified ITR does not contain any nucleotidedeletions in the RBE-containing portion of the A or A′ regions, so asnot to interfere with DNA replication (e.g. binding to an RBE by Repprotein, or nicking at a terminal resolution site). In some embodiments,a modified ITR encompassed for use herein has one or more deletions inthe B, B′, C, and/or C region as described herein.

In some embodiments, a ceDNA vector for expression of FVIII proteincomprising a symmetric ITR pair or asymmetric ITR pair comprises aregulatory switch as disclosed herein and at least one modified ITRselected having the nucleotide sequence selected from any of the groupconsisting of: SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187.

In another embodiment, the structure of the structural element can bemodified. For example, the structural element a change in the height ofthe stem and/or the number of nucleotides in the loop. For example, theheight of the stem can be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides ormore or any range therein. In one embodiment, the stem height can beabout 5 nucleotides to about 9 nucleotides and functionally interactswith Rep. In another embodiment, the stem height can be about 7nucleotides and functionally interacts with Rep. In another example, theloop can have 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more or anyrange therein.

In another embodiment, the number of GAGY binding sites or GAGY-relatedbinding sites within the RBE or extended RBE can be increased ordecreased. In one example, the RBE or extended RBE, can comprise 1, 2,3, 4, 5, or 6 or more GAGY binding sites or any range therein. Each GAGYbinding site can independently be an exact GAGY sequence or a sequencesimilar to GAGY as long as the sequence is sufficient to bind a Repprotein.

In another embodiment, the spacing between two elements (such as but notlimited to the RBE and a hairpin) can be altered (e.g., increased ordecreased) to alter functional interaction with a large Rep protein. Forexample, the spacing can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more or any rangetherein.

The ceDNA vector for expression of FVIII protein as described herein caninclude an ITR structure that is modified with respect to the wild typeAAV2 ITR structure disclosed herein, but still retains an operable RBE,TRS and RBE′ portion. FIG. 2A and FIG. 2B show one possible mechanismfor the operation of a TRS site within a wild type ITR structure portionof a ceDNA vector for expression of FVIII protein. In some embodiments,the ceDNA vector for expression of FVIII protein contains one or morefunctional ITR polynucleotide sequences that comprise a Rep-binding site(RBS; 5′-GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60) for AAV2) and a terminalresolution site (TRS; 5′-AGTT (SEQ ID NO: 62)). In some embodiments, atleast one ITR (wt or modified ITR) is functional. In alternativeembodiments, where a ceDNA vector for expression of FVIII proteincomprises two modified ITRs that are different or asymmetrical to eachother, at least one modified ITR is functional and at least one modifiedITR is non-functional.

In some embodiments, the modified ITR (e.g., the left or right ITR) of aceDNA vector for expression of FVIII protein as described herein hasmodifications within the loop arm, the truncated arm, or the spacer.Exemplary sequences of ITRs having modifications within the loop arm,the truncated arm, or the spacer are listed in Table 2 (i.e., SEQ IDNOS: 135-190, 200-233); Table 3 (e.g., SEQ ID Nos: 234-263); Table 4(e.g., SEQ ID NOs: 264-293); Table 5 (e.g., SEQ ID Nos: 294-318 herein);Table 6 (e.g., SEQ ID NO: 319-468; and Tables 7-9 (e.g., SEQ ID Nos:101-110, 111-112, 115-134) or Table 10A or 10B (e.g., SEQ ID Nos: 9,100, 469-483, 484-499) of International application PCT/US18/49996,which is incorporated herein in its entirety by reference.

In some embodiments, the modified ITR for use in a ceDNA vector forexpression of FVIII protein comprising an asymmetric ITR pair, orsymmetric mod-ITR pair is selected from any or a combination of thoseshown in Tables 2, 3, 4, 5, 6, 7, 8, 9 and 10A-10B of Internationalapplication PCT/US18/49996 which is incorporated herein in its entiretyby reference.

Additional exemplary modified ITRs for use in a ceDNA vector forexpression of FVIII protein comprising an asymmetric ITR pair, orsymmetric mod-ITR pair in each of the above classes are provided inTables 5A and 5B. The predicted secondary structure of the Rightmodified ITRs in Table 5A are shown in FIG. 7A of InternationalApplication PCT/US2018/064242, filed Dec. 6, 2018, and the predictedsecondary structure of the Left modified ITRs in Table 5B are shown inFIG. 7B of International Application PCT/US2018/064242, filed Dec. 6,2018, which is incorporated herein in its entirety by reference.

Table 5A and Table 5B show exemplary right and left modified ITRs.

Table 5A: Exemplary modified right ITRs. These exemplary modified rightITRs can comprise the RBE of GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60), spacerof ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO:70) and RBE′ (i.e., complement to RBE) of GAGCGAGCGAGCGCGC (SEQ ID NO:71).

TABLE 5A Exemplary Right modified ITRs ITR SEQ ID Construct Sequence NO:ITR-18 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 15 RightCTCGCTCACTGAGGCGCACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-19AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 16 RightCTCGCTCACTGAGGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-20AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 17 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-21AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 18 RightCTCGCTCACTGAGGCTTTGCCTCAGTGAGCGAGCGAGCGCGCAGC TGCCTGCAGG ITR-22AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 19 RightCTCGCTCACTGAGGCCGGGCGACAAAGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGC AGG ITR-23AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 20 RightCTCGCTCACTGAGGCCGGGCGAAAATCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAG G ITR-24AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 21 RightCTCGCTCACTGAGGCCGGGCGAAACGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-25AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 22 RightCTCGCTCACTGAGGCCGGGCAAAGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-26AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 23 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGC AGG ITR-27AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 24 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGTTTCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAG G ITR-28AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 25 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGTTTCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-29AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 26 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCTTTGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-30AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 27 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCTTTGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-31AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 28 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCTTTGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-32AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 29 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGTTTCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-49AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 30 RightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG ITR-50AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG 31 rightCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

TABLE 5B: Exemplary modified left ITRs. These exemplary modified leftITRs can comprise the RBE of GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60), spacerof ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO:70) and RBE complement (RBE′) of GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

TABLE 5B Exemplary modified left ITRs ITR-33CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 32 LeftAAACCCGGGCGTGCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-34CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGTCGGGC 33 LeftGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-35CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 34 LeftCAAAGCCCGGGCGTCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-36CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCGCCCGGGC 35 LeftGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-37CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCAAAGCCTC 36 LeftAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCT ITR-38CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 37 LeftCAAAGCCCGGGCGTCGGGCGACTTTGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGT TCCT ITR-39CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 38 LeftCAAAGCCCGGGCGTCGGGCGATTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CT ITR-40CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 39 LeftCAAAGCCCGGGCGTCGGGCGTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-41CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 40 LeftCAAAGCCCGGGCGTCGGGCTTTGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-42CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG 41 LeftAAACCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGT TCCT ITR-43CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGA 42 LeftAACCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CT ITR-44CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGAA 43 LeftACGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-45CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCAAA 44 LeftGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-46CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCAAAG 45 LeftGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-47CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCAAAGC 46 LeftGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT ITR-48CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGAAACGT 47 LeftCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

In one embodiment, a ceDNA vector for expression of FVIII proteincomprises, in the 5′ to 3′ direction: a first adeno-associated virus(AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest(for example an expression cassette as described herein) and a secondAAV ITR, where the first ITR (5′ ITR) and the second ITR (3′ ITR) areasymmetric with respect to each other—that is, they have a different3D-spatial configuration from one another. As an exemplary embodiment,the first ITR can be a wild-type ITR and the second ITR can be a mutatedor modified ITR, or vice versa, where the first ITR can be a mutated ormodified ITR and the second ITR a wild-type ITR. In some embodiment, thefirst ITR and the second ITR are both mod-ITRs, but have differentsequences, or have different modifications, and thus are not the samemodified ITRs, and have different 3D spatial configurations. Stateddifferently, a ceDNA vector with asymmetric ITRs comprises ITRs whereany changes in one ITR relative to the WT-ITR are not reflected in theother ITR; or alternatively, where the asymmetric ITRs have a modifiedasymmetric ITR pair can have a different sequence and differentthree-dimensional shape with respect to each other. Exemplary asymmetricITRs in the ceDNA vector for expression of FVIII protein and for use togenerate a ceDNA-plasmid are shown in Table 5A and 5B.

In an alternative embodiment, a ceDNA vector for expression of FVIIIprotein comprises two symmetrical mod-ITRs—that is, both ITRs have thesame sequence, but are reverse complements (inverted) of each other. Insome embodiments, a symmetrical mod-ITR pair comprises at least one orany combination of a deletion, insertion, or substitution relative towild type ITR sequence from the same AAV serotype. The additions,deletions, or substitutions in the symmetrical ITR are the same but thereverse complement of each other. For example, an insertion of 3nucleotides in the C region of the 5′ ITR would be reflected in theinsertion of 3 reverse complement nucleotides in the correspondingsection in the C′ region of the 3′ ITR. Solely for illustration purposesonly, if the addition is AACG in the 5′ ITR, the addition is CGTT in the3′ ITR at the corresponding site. For example, if the 5′ ITR sensestrand is ATCGATCG with an addition of AACG between the G and A toresult in the sequence ATCGAACGATCG (SEQ ID NO: 51). The corresponding3′ ITR sense strand is CGATCGAT (the reverse complement of ATCGATCG)with an addition of CGTT (i.e. the reverse complement of AACG) betweenthe T and C to result in the sequence CGATCGTTCGAT (SEQ ID NO: 49) (thereverse complement of ATCGAACGATCG) (SEQ ID NO: 51).

In alternative embodiments, the modified ITR pair are substantiallysymmetrical as defined herein—that is, the modified ITR pair can have adifferent sequence but have corresponding or the same symmetricalthree-dimensional shape. For example, one modified ITR can be from oneserotype and the other modified ITR be from a different serotype, butthey have the same mutation (e.g., nucleotide insertion, deletion orsubstitution) in the same region. Stated differently, for illustrativepurposes only, a 5′ mod-ITR can be from AAV2 and have a deletion in theC region, and the 3′ mod-ITR can be from AAV5 and have the correspondingdeletion in the C′ region, and provided the 5′ mod-ITR and the 3′mod-ITR have the same or symmetrical three-dimensional spatialorganization, they are encompassed for use herein as a modified ITRpair.

In some embodiments, a substantially symmetrical mod-ITR pair has thesame A, C-C′ and B-B′ loops in 3D space, e.g., if a modified ITR in asubstantially symmetrical mod-ITR pair has a deletion of a C-C′ arm,then the cognate mod-ITR has the corresponding deletion of the C-C′ loopand also has a similar 3D structure of the remaining A and B-B′ loops inthe same shape in geometric space of its cognate mod-ITR. By way ofexample only, substantially symmetrical ITRs can have a symmetricalspatial organization such that their structure is the same shape ingeometrical space. This can occur, e.g., when a G-C pair is modified,for example, to a C-G pair or vice versa, or A-T pair is modified to aT-A pair, or vice versa. Therefore, using the exemplary example above ofmodified 5′ ITR as a ATCGAACGATCG (SEQ ID NO: 51), and modified 3′ ITRas CGATCGTTCGAT (SEQ ID NO: 49) (i.e., the reverse complement ofATCGAACGATCG (SEQ ID NO: 51)), these modified ITRs would still besymmetrical if, for example, the 5′ ITR had the sequence of ATCGAACCATCG(SEQ ID NO: 50), where G in the addition is modified to C, and thesubstantially symmetrical 3′ ITR has the sequence of CGATCGTTCGAT (SEQID NO: 49), without the corresponding modification of the T in theaddition to a. In some embodiments, such a modified ITR pair aresubstantially symmetrical as the modified ITR pair has symmetricalstereochemistry.

Table 6 shows exemplary symmetric modified ITR pairs (i.e. a leftmodified ITRs and the symmetric right modified ITR) for use in a ceDNAvector for expression of FVIII protein. The bold (red) portion of thesequences identify partial ITR sequences (i.e., sequences of A-A′, C-C′and B-B′ loops), also shown in FIGS. 31A-46B. These exemplary modifiedITRs can comprise the RBE of GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60), spacerof ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO:70) and RBE′ (i.e., complement to RBE) of GAGCGAGCGAGCGCGC (SEQ ID NO:71).

TABLE 6Exemplary symmetric modified ITR pairs in a ceDNA vector for expression ofFVIII protein LEFT modified ITR Symmetric RIGHT modified ITR(modified 5′ ITR) (modified 3′ ITR) SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 15 AGGAACCCCTAGTGATG NO: 32 GCTCGCTCACTGAGGCCGCC(ITR-18, right) GAGTTGGCCACTCCCTCT (ITR-33 CGGGAAACCCGGGCGTGCGCCTGCGCGCTCGCTCGC left) CTCAGTGAGCGAGCGAGCGC TCACTGAGGCGCACGCGCAGAGAGGGAGTGGCCAACT CCGGGTTTCCCGGGCG CCATCACTAGGGGTTCCTGCCTCAGTGAGCGAGC GAGCGCGCAGCTGCCT GCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 48 AGGAACCCCTAGTGATG NO: 33 GCTCGCTCACTGAGGCCGTC(ITR-51, right) GAGTTGGCCACTCCCTCT (ITR-34 GGGCGACCTTTGGTCGCCCGCTGCGCGCTCGCTCGC left) GCCTCAGTGAGCGAGCGAGC TCACTGAGGCCGGGCGGCGCAGAGAGGGAGTGGCCA ACCAAAGGTCGCCCGA ACTCCATCACTAGGGGTTCCTCGGCCTCAGTGAGCGA GCGAGCGCGCAGCTGC CTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 16 AGGAACCCCTAGTGATG NO: 34 GCTCGCTCACTGAGGCCGCC(ITR-19, right) GAGTTGGCCACTCCCTCT (ITR-35 CGGGCAAAGCCCGGGCGTCGCTGCGCGCTCGCTCGC left) GCCTCAGTGAGCGAGCGAGC TCACTGAGGCCGACGCGCGCAGAGAGGGAGTGGCCA CCGGGCTTTGCCCGGG ACTCCATCACTAGGGGTTCCTCGGCCTCAGTGAGCGA GCGAGCGCGCAGCTGC CTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 17 AGGAACCCCTAGTGATG NO: 35 GCTCGCTCACTGAGGCGCCC(ITR-20, right) GAGTTGGCCACTCCCTCT (ITR-36 GGGCGTCGGGCGACCTTTGGCTGCGCGCTCGCTCGC left) TCGCCCGGCCTCAGTGAGCG TCACTGAGGCCGGGCGAGCGAGCGCGCAGAGAGGGA ACCAAAGGTCGCCCGA GTGGCCAACTCCATCACTAGGCGCCCGGGCGCCTCAG GGTTCCT TGAGCGAGCGAGCGCG CAGCTGCCTGCAGG SEQ IDCCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 18 AGGAACCCCTAGTGATG NO: 36GCTCGCTCACTGAGGCAAAG (ITR-21, right) GAGTTGGCCACTCCCTCT (ITR-37CCTCAGTGAGCGAGCGAGCG CTGCGCGCTCGCTCGC left) CGCAGAGAGGGAGTGGCCAACTCACTGAGGCTTTGCC TCCATCACTAGGGGTTCCT TCAGTGAGCGAGCGAG CGCGCAGCTGCCTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 19 AGGAACCCCTAGTGATG NO: 37GCTCGCTCACTGAGGCCGCC (ITR-22 right) GAGTTGGCCACTCCCTCT (ITR-38CGGGCAAAGCCCGGGCGTCG CTGCGCGCTCGCTCGC left) GGCGACTTTGTCGCCCGGCCTCACTGAGGCCGGGCG TCAGTGAGCGAGCGAGCGCG ACAAAGTCGCCCGACGCAGAGAGGGAGTGGCCAACTC CCCGGGCTTTGCCCGG CATCACTAGGGGTTCCTGCGGCCTCAGTGAGCG AGCGAGCGCGCAGCTG CCTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 20 AGGAACCCCTAGTGATG NO: 38 GCTCGCTCACTGAGGCCGCC(ITR-23, right) GAGTTGGCCACTCCCTCT (ITR-39 CGGGCAAAGCCCGGGCGTCGCTGCGCGCTCGCTCGC left) GGCGATTTTCGCCCGGCCTC TCACTGAGGCCGGGCGAGTGAGCGAGCGAGCGCGCA AAAATCGCCCGACGCC GAGAGGGAGTGGCCAACTCCACGGGCTTTGCCCGGGC TCACTAGGGGTTCCT GGCCTCAGTGAGCGAG CGAGCGCGCAGCTGCCTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 21 AGGAACCCCTAGTGATGNO: 39 GCTCGCTCACTGAGGCCGCC (ITR-24, right) GAGTTGGCCACTCCCTCT (ITR-40CGGGCAAAGCCCGGGCGTCG CTGCGCGCTCGCTCGC left) GGCGTTTCGCCCGGCCTCAGTCACTGAGGCCGGGCG TGAGCGAGCGAGCGCGCAGA AAACGCCCGACGCCCGGAGGGAGTGGCCAACTCCATC GGCTTTGCCCGGGCGG ACTAGGGGTTCCT CCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGC AGG SEQ ID CCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 22AGGAACCCCTAGTGATG NO: 40 GCTCGCTCACTGAGGCCGCC (ITR-25 right)GAGTTGGCCACTCCCTCT (ITR-41 CGGGCAAAGCCCGGGCGTCG CTGCGCGCTCGCTCGC left)GGCTTTGCCCGGCCTCAGTG TCACTGAGGCCGGGCA AGCGAGCGAGCGCGCAGAGAAAGCCCGACGCCCGGG GGGAGTGGCCAACTCCATCAC CTTTGCCCGGGCGGCC TAGGGGTTCCTTCAGTGAGCGAGCGAG CGCGCAGCTGCCTGCAG G SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 23 AGGAACCCCTAGTGATG NO: 41 GCTCGCTCACTGAGGCCGCC(ITR-26 right) GAGTTGGCCACTCCCTCT (ITR-42 CGGGAAACCCGGGCGTCGGGCTGCGCGCTCGCTCGC left) CGACCTTTGGTCGCCCGGCC TCACTGAGGCCGGGCGTCAGTGAGCGAGCGAGCGCG ACCAAAGGTCGCCCGA CAGAGAGGGAGTGGCCAACTCCGCCCGGGTTTCCCGG CATCACTAGGGGTTCCT GCGGCCTCAGTGAGCG AGCGAGCGCGCAGCTGCCTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 24 AGGAACCCCTAGTGATGNO: 42 GCTCGCTCACTGAGGCCGCC (ITR-27 right) GAGTTGGCCACTCCCTCT (ITR-43CGGAAACCGGGCGTCGGGCG CTGCGCGCTCGCTCGC left) ACCTTTGGTCGCCCGGCCTCTCACTGAGGCCGGGCG AGTGAGCGAGCGAGCGCGCA ACCAAAGGTCGCCCGAGAGAGGGAGTGGCCAACTCCA CGCCCGGTTTCCGGGC TCACTAGGGGTTCCT GGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCC TGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 25AGGAACCCCTAGTGATG NO: 43 GCTCGCTCACTGAGGCCGCC (ITR-28 right)GAGTTGGCCACTCCCTCT (ITR-44 CGAAACGGGCGTCGGGCGAC CTGCGCGCTCGCTCGCCTTTGGTCGCCCGGCCTCAG TCACTGAGGCCGGGCG left) TGAGCGAGCGAGCGCGCAGAACCAAAGGTCGCCCGA GAGGGAGTGGCCAACTCCATC CGCCCGTTTCGGGCGG ACTAGGGGTTCCTCCTCAGTGAGCGAGCG AGCGCGCAGCTGCCTGC AGG SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 26 AGGAACCCCTAGTGATG NO: 44 GCTCGCTCACTGAGGCCGCC(ITR-29, right) GAGTTGGCCACTCCCTCT (ITR-45 CAAAGGGCGTCGGGCGACCTCTGCGCGCTCGCTCGC left) TTGGTCGCCCGGCCTCAGTG TCACTGAGGCCGGGCGAGCGAGCGAGCGCGCAGAGA ACCAAAGGTCGCCCGA GGGAGTGGCCAACTCCATCACCGCCCTTTGGGCGGCC TAGGGGTTCCT TCAGTGAGCGAGCGAG CGCGCAGCTGCCTGCAG G SEQ IDCCTGCAGGCAGCTGCGCGCTC SEQ ID NO: AGGAACCCCTAGTGATG NO: 45GCTCGCTCACTGAGGCCGCC 27(ITR-30, right) GAGTTGGCCACTCCCTCT (ITR-46AAAGGCGTCGGGCGACCTTT CTGCGCGCTCGCTCGC left) GGTCGCCCGGCCTCAGTGAGTCACTGAGGCCGGGCG CGAGCGAGCGCGCAGAGAGG ACCAAAGGTCGCCCGAGAGTGGCCAACTCCATCACTA CGCCTTTGGCGGCCTC GGGGTTCCT AGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTC SEQ ID NO: 28AGGAACCCCTAGTGATG NO: 46 GCTCGCTCACTGAGGCCGCA (ITR-31, right)GAGTTGGCCACTCCCTCT (ITR-47, AAGCGTCGGGCGACCTTTGG CTGCGCGCTCGCTCGC left)TCGCCCGGCCTCAGTGAGCG TCACTGAGGCCGGGCG AGCGAGCGCGCAGAGAGGGAACCAAAGGTCGCCCGA GTGGCCAACTCCATCACTAGG CGCTTTGCGGCCTCAG GGTTCCTTGAGCGAGCGAGCGCG CAGCTGCCTGCAGG SEQ ID CCTGCAGGCAGCTGCGCGCTCSEQ ID NO: 29 AGGAACCCCTAGTGATG NO: 47 GCTCGCTCACTGAGGCCGAA(ITR-32 right) GAGTTGGCCACTCCCTCT (ITR-48, ACGTCGGGCGACCTTTGGTCCTGCGCGCTCGCTCGC left) GCCCGGCCTCAGTGAGCGAG TCACTGAGGCCGGGCGCGAGCGCGCAGAGAGGGAGT ACCAAAGGTCGCCCGA GGCCAACTCCATCACTAGGGGCGTTTCGGCCTCAGTG TTCCT AGCGAGCGAGCGCGCA GCTGCCTGCAGG

In some embodiments, a ceDNA vector for expression of FVIII proteincomprising an asymmetric ITR pair can comprise an ITR with amodification corresponding to any of the modifications in ITR sequencesor ITR partial sequences shown in any one or more of Tables 5A-5Bherein, or the sequences shown in FIG. 7A-7B of InternationalApplication PCT/US2018/064242, filed Dec. 6, 2018, which is incorporatedherein in its entirety, or disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9 or10A-10B of International application PCT/US18/49996 filed Sep. 7, 2018which is incorporated herein in its entirety by reference.

V. Exemplary ceDNA Vectors

As described above, the present disclosure relates to recombinant ceDNAexpression vectors and ceDNA vectors that encode FVIII protein,comprising any one of: an asymmetrical ITR pair, a symmetrical ITR pair,or substantially symmetrical ITR pair as described above. In certainembodiments, the disclosure relates to recombinant ceDNA vectors forexpression of FVIII protein having flanking ITR sequences and atransgene, where the ITR sequences are asymmetrical, symmetrical orsubstantially symmetrical relative to each other as defined herein, andthe ceDNA further comprises a nucleotide sequence of interest (forexample an expression cassette comprising the nucleic acid of atransgene) located between the flanking ITRs, wherein said nucleic acidmolecule is devoid of viral capsid protein coding sequences.

The ceDNA expression vector for expression of FVIII protein may be anyceDNA vector that can be conveniently subjected to recombinant DNAprocedures including nucleotide sequence(s) as described herein,provided at least one ITR is altered. The ceDNA vectors for expressionof FVIII protein of the present disclosure are compatible with the hostcell into which the ceDNA vector is to be introduced. In certainembodiments, the ceDNA vectors may be linear. In certain embodiments,the ceDNA vectors may exist as an extrachromosomal entity. In certainembodiments, the ceDNA vectors of the present disclosure may contain anelement(s) that permits integration of a donor sequence into the hostcell's genome. As used herein “transgene” and “heterologous nucleotidesequence” are synonymous, and encode FVIII protein, as described herein.

Referring now to FIGS. 1A-1G, schematics of the functional components oftwo non-limiting plasmids useful in making a ceDNA vector for expressionof FVIII protein are shown. FIG. 1A, 1B, 1D, 1F show the construct ofceDNA vectors or the corresponding sequences of ceDNA plasmids forexpression of FVIII protein. ceDNA vectors are capsid-free and can beobtained from a plasmid encoding in this order: a first ITR, anexpressible transgene cassette and a second ITR, where the first andsecond ITR sequences are asymmetrical, symmetrical or substantiallysymmetrical relative to each other as defined herein. ceDNA vectors forexpression of FVIII protein are capsid-free and can be obtained from aplasmid encoding in this order: a first ITR, an expressible transgene(protein or nucleic acid) and a second ITR, where the first and secondITR sequences are asymmetrical, symmetrical or substantially symmetricalrelative to each other as defined herein. In some embodiments, theexpressible transgene cassette includes, as needed: anenhancer/promoter, one or more homology arms, a donor sequence, apost-transcription regulatory element (e.g., WPRE, e.g., SEQ ID NO:67)), and a polyadenylation and termination signal (e.g., BGH polyA,e.g., SEQ ID NO: 68).

FIG. 5 is a gel confirming the production of ceDNA from multiple plasmidconstructs using the method described in the Examples. The ceDNA isconfirmed by a characteristic band pattern in the gel, as discussed withrespect to FIG. 4A above and in the Examples.

A. Regulatory Elements

The ceDNA vectors for expression of FVIII protein as described hereincomprising an asymmetric ITR pair or symmetric ITR pair as definedherein, can further comprise a specific combination of cis-regulatoryelements. The cis-regulatory elements include, but are not limited to, apromoter, a riboswitch, an insulator, a mir-regulatable element, apost-transcriptional regulatory element, a tissue- and celltype-specific promoter and an enhancer. In some embodiments, the ITR canact as the promoter for the transgene, e.g., FVIII protein. In someembodiments, the ceDNA vector for expression of FVIII protein asdescribed herein comprises additional components to regulate expressionof the transgene, for example, regulatory switches as described herein,to regulate the expression of the transgene, or a kill switch, which cankill a cell comprising the ceDNA vector encoding FVIII protein thereof.Regulatory elements, including Regulatory Switches that can be used inthe present invention are more fully discussed in Internationalapplication PCT/US18/49996, which is incorporated herein in its entiretyby reference.

In embodiments, the second nucleotide sequence includes a regulatorysequence, and a nucleotide sequence encoding a nuclease. In certainembodiments the gene regulatory sequence is operably linked to thenucleotide sequence encoding the nuclease. In certain embodiments, theregulatory sequence is suitable for controlling the expression of thenuclease in a host cell. In certain embodiments, the regulatory sequenceincludes a suitable promoter sequence, being able to directtranscription of a gene operably linked to the promoter sequence, suchas a nucleotide sequence encoding the nuclease(s) of the presentdisclosure. In certain embodiments, the second nucleotide sequenceincludes an intron sequence linked to the 5′ terminus of the nucleotidesequence encoding the nuclease. In certain embodiments, an enhancersequence is provided upstream of the promoter to increase the efficacyof the promoter. In certain embodiments, the regulatory sequenceincludes an enhancer and a promoter, wherein the second nucleotidesequence includes an intron sequence upstream of the nucleotide sequenceencoding a nuclease, wherein the intron includes one or more nucleasecleavage site(s), and wherein the promoter is operably linked to thenucleotide sequence encoding the nuclease.

The ceDNA vectors for expression of FVIII protein producedsynthetically, or using a cell-based production method as describedherein in the Examples, can further comprise a specific combination ofcis-regulatory elements such as WHP posttranscriptional regulatoryelement (WPRE) (e.g., SEQ ID NO: 67) and BGH polyA (SEQ ID NO: 68).Suitable expression cassettes for use in expression constructs are notlimited by the packaging constraint imposed by the viral capsid.

(i) Promoters:

It will be appreciated by one of ordinary skill in the art thatpromoters used in the ceDNA vectors for expression of FVIII protein asdisclosed herein should be tailored as appropriate for the specificsequences they are promoting. According to some embodiments, thepromoter is any promoter or promoter sequence set forth in InternationalApplication No. PCT/US2020/021328, filed on Mar. 6, 2020, incorporatedby reference in its entirety herein.

According to some embodiments, the promoter is VandenDriessche (VD)promoter. According to some embodiments, the VD promoter comprises SEQID NO: 191 shown below:

(SEQ ID NO: 191) CGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTG.

According to some embodiments, the promoter comprises a nucleic acidsequence at least about 85% identical to SEQ ID NO: 191. According tosome embodiments, the promoter comprises a nucleic acid sequence atleast about 90% identical to SEQ ID NO: 191. According to someembodiments, the promoter comprises a nucleic acid sequence at leastabout 95% identical to SEQ ID NO: 191. According to some embodiments,the promoter comprises a nucleic acid sequence at least about 96%identical to SEQ ID NO: 191. According to some embodiments, the promotercomprises a nucleic acid sequence at least about 97% identical to SEQ IDNO: 191. According to some embodiments, the promoter comprises a nucleicacid sequence at least about 98% identical to SEQ ID NO: 191. Accordingto some embodiments, the promoter comprises a nucleic acid sequence atleast about 99% identical to SEQ ID NO: 191. According to someembodiments, the promoter consists of the nucleic acid sequence of SEQID NO: 191.

Expression cassettes of the ceDNA vector for expression of FVIII proteincan include a promoter, which can influence overall expression levels aswell as cell-specificity. For transgene expression, e.g., expression ofFVIII protein, they can include a highly active virus-derived immediateearly promoter. Expression cassettes can contain tissue-specificeukaryotic promoters to limit transgene expression to specific celltypes and reduce toxic effects and immune responses resulting fromunregulated, ectopic expression. In some embodiments, an expressioncassette can contain a promoter or synthetic regulatory element, such asa CAG promoter (SEQ ID NO: 72). The CAG promoter comprises (i) thecytomegalovirus (CMV) early enhancer element, (ii) the promoter, thefirst exon and the first intron of chicken beta-actin gene, and (iii)the splice acceptor of the rabbit beta-globin gene. Alternatively, anexpression cassette can contain an Alpha-1-antitrypsin (AAT) promoter(SEQ ID NO: 73 or SEQ ID NO: 74), a liver specific (LP1) promoter (SEQID NO: 75 or SEQ ID NO: 76), or a Human elongation factor-1 alpha (EF1a)promoter (e.g., SEQ ID NO: 77 or SEQ ID NO: 78). In some embodiments,the expression cassette includes one or more constitutive promoters, forexample, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionallywith the RSV enhancer), or a cytomegalovirus (CMV) immediate earlypromoter (optionally with the CMV enhancer, e.g., SEQ ID NO: 79).Alternatively, an inducible promoter, a native promoter for a transgene,a tissue-specific promoter, or various promoters known in the art can beused.

Suitable promoters, including those described herein, can be derivedfrom viruses and can therefore be referred to as viral promoters, orthey can be derived from any organism, including prokaryotic oreukaryotic organisms. Suitable promoters can be used to drive expressionby any RNA polymerase (e.g., pol I, pol II, pol III). Exemplarypromoters include, but are not limited to the SV40 early promoter, mousemammary tumor virus long terminal repeat (LTR) promoter; adenovirusmajor late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, acytomegalovirus (CMV) promoter such as the CMV immediate early promoterregion (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 smallnuclear promoter (U6, e.g., SEQ ID NO: 80) (Miyagishi et al., NatureBiotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia etal., Nucleic Acids Res. 2003 Sep. 1; 31(17)), a human H1 promoter (H1)(e.g., SEQ ID NO: 81 or SEQ ID NO: 155), a CAG promoter, a human alpha1-antitypsin (HAAT) promoter (e.g., SEQ ID NO: 82), and the like. Incertain embodiments, these promoters are altered at their downstreamintron containing end to include one or more nuclease cleavage sites. Incertain embodiments, the DNA containing the nuclease cleavage site(s) isforeign to the promoter DNA.

In one embodiment, the promoter used is the native promoter of the geneencoding the therapeutic protein. The promoters and other regulatorysequences for the respective genes encoding the therapeutic proteins areknown and have been characterized. The promoter region used may furtherinclude one or more additional regulatory sequences (e.g., native),e.g., enhancers, (e.g. SEQ ID NO: 79 and SEQ ID NO: 83), including aSV40 enhancer (SEQ ID NO: 126).

In some embodiments, a promoter may also be a promoter from a human genesuch as human ubiquitin C (hUbC), human actin, human myosin, humanhemoglobin, human muscle creatine, or human metallothionein. Thepromoter may also be a tissue specific promoter, such as a liverspecific promoter, such as human alpha 1-antitypsin (HAAT), natural orsynthetic. In one embodiment, delivery to the liver can be achievedusing endogenous ApoE specific targeting of the composition comprising aceDNA vector to hepatocytes via the low-density lipoprotein (LDL)receptor present on the surface of the hepatocyte.

Non-limiting examples of suitable promoters for use in accordance withthe present invention include any of the promoters described herein, orany of the following: the CAG promoter of, for example (SEQ ID NO: 72),the HAAT promoter (SEQ ID NO: 82), the human EF1-α promoter (SEQ ID NO:77) or a fragment of the EF1a promoter (SEQ ID NO: 78), 1E2 promoter(e.g., SEQ ID NO: 84) and the rat EF1-α promoter (SEQ ID NO: 85), mEF1promoter (SEQ ID NO: 59), or 1E1 promoter fragment (SEQ ID NO: 125).

(ii) Enhancers

In some embodiments, a ceDNA expressing FVIII comprises one or moreenhancers. In some embodiments, an enhancer sequence is located 5′ ofthe promoter sequence. In some embodiments, the enhancer sequence islocated 3′ of the promoter sequence. According to some embodiments, theenhancer is any enhancer or enhancer sequence set forth in InternationalApplication No. PCT/US2020/021328, filed on Mar. 6, 2020, incorporatedby reference in its entirety herein.

(iii) 5′ UTR Sequences and Intron Sequences

In some embodiments, a ceDNA vector comprises a 5′ UTR sequence and/oran intron sequence that located 3′ of the 5′ ITR sequence. In someembodiments, the 5′ UTR is located 5′ of the transgene, e.g., sequenceencoding the FVIII protein. Exemplary 5′ UTR sequences listed inInternational Application No. PCT/US2020/021328, for example in Table9A, incorporated by reference in its entirety herein.

(iv) 3′ UTR Sequences

In some embodiments, a ceDNA vector comprises a 3′ UTR sequence thatlocated 5′ of the 3′ ITR sequence. In some embodiments, the 3′ UTR islocated 3′ of the transgene, e.g., sequence encoding the FVIII protein.Exemplary 3′ UTR sequences listed in International Application No.PCT/US2020/021328, for example in Table 9B, incorporated by reference inits entirety herein. (v)

Polyadenylation Sequences:

A sequence encoding a polyadenylation sequence can be included in theceDNA vector for expression of FVIII protein to stabilize an mRNAexpressed from the ceDNA vector, and to aid in nuclear export andtranslation. In one embodiment, the ceDNA vector does not include apolyadenylation sequence. In other embodiments, the ceDNA vector forexpression of FVIII protein includes at least 1, at least 2, at least 3,at least 4, at least 5, at least 10, at least 15, at least 20, at least25, at least 30, at least 40, least 45, at least 50 or more adeninedinucleotides. In some embodiments, the polyadenylation sequencecomprises about 43 nucleotides, about 40-50 nucleotides, about 40-55nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or anyrange there between.

The expression cassettes can include any poly-adenylation sequence knownin the art or a variation thereof. In some embodiments, apoly-adenylation (polyA) sequence is selected from any of those listedin International Application No. PCT/US2020/021328, for example in Table10, incorporated by reference in its entirety herein. Other polyAsequences commonly known in the art can also be used, e.g., includingbut not limited to, naturally occurring sequence isolated from bovineBGHpA (e.g., SEQ ID NO: 68) or a virus SV40pA (e.g., SEQ ID NO: 86), ora synthetic sequence (e.g., SEQ ID NO: 87). Some expression cassettescan also include SV40 late polyA signal upstream enhancer (USE)sequence. In some embodiments, a USE sequence can be used in combinationwith SV40pA or heterologous poly-A signal. PolyA sequences are located3′ of the transgene encoding the FVIII protein.

The expression cassettes can also include a post-transcriptional elementto increase the expression of a transgene. In some embodiments,Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element(WPRE) (e.g., SEQ ID NO: 67) is used to increase the expression of atransgene. Other posttranscriptional processing elements such as thepost-transcriptional element from the thymidine kinase gene of herpessimplex virus, or hepatitis B virus (HBV) can be used. Secretorysequences can be linked to the transgenes, e.g., VH-02 and VK-A26sequences, e.g., SEQ ID NO: 88 and SEQ ID NO: 89.

(vi) Nuclear Localization Sequences

In some embodiments, the ceDNA vector for expression of FVIII proteincomprises one or more nuclear localization sequences (NLSs), forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In someembodiments, the one or more NLSs are located at or near theamino-terminus, at or near the carboxy-terminus, or a combination ofthese (e.g., one or more NLS at the amino-terminus and/or one or moreNLS at the carboxy terminus). When more than one NLS is present, eachcan be selected independently of the others, such that a single NLS ispresent in more than one copy and/or in combination with one or moreother NLSs present in one or more copies. Non-limiting examples of NLSsare shown in Table 7.

TABLE 7 Nuclear Localization Signals SEQ ID SOURCE SEQUENCE NO.SV40 virus large PKKKRKV (encoded by CCCAAGAAGAAGAGGAAGGTG;  90T-antigen SEQ ID NO: 91) nucleoplasmin KRPAATKKAGQAKKKK  92 c-mycPAAKRVKLD  93 RQRRNELKRSP 94 hRNPA1 M9NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY  95 IBB domain fromRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV 96 importin-alphamyoma T protein VSRKRPRP  97 PPKKARED  98 human p53 PQPKKKPL  99mouse c-abl IV SALIKKKKKMAP 100 influenza virus DRLRR 117 NS1 PKQKKRK118 Hepatitis virus RKLKKKIKKL 119 delta antigen mouse Mx1 proteinREKKKFLKRR 120 human poly (ADP- KRKGDEVDGVDEVAKKKSKK 121ribose) polymerase steroid hormone RKCLQAGMNLEARKTKK 122receptors (human) glucocorticoid

B. Additional Components of ceDNA Vectors

The ceDNA vectors for expression of FVIII protein of the presentdisclosure may contain nucleotides that encode other components for geneexpression. For example, to select for specific gene targeting events, aprotective shRNA may be embedded in a microRNA and inserted into arecombinant ceDNA vector designed to integrate site-specifically intothe highly active locus, such as an albumin locus. Such embodiments mayprovide a system for in vivo selection and expansion of gene-modifiedhepatocytes in any genetic background such as described in Nygaard etal., A universal system to select gene-modified hepatocytes in vivo,Gene Therapy, Jun. 8, 2016. The ceDNA vectors of the present disclosuremay contain one or more selectable markers that permit selection oftransformed, transfected, transduced, or the like cells. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, NeoR,and the like. In certain embodiments, positive selection markers areincorporated into the donor sequences such as NeoR. Negative selectionsmarkers may be incorporated downstream the donor sequences, for examplea nucleic acid sequence HSV-tk encoding a negative selection marker maybe incorporated into a nucleic acid construct downstream the donorsequence.

C. Regulatory Switches

A molecular regulatory switch is one which generates a measurable changein state in response to a signal. Such regulatory switches can beusefully combined with the ceDNA vectors for expression of FVIII proteinas described herein to control the output of expression of FVIII proteinfrom the ceDNA vector. In some embodiments, the ceDNA vector forexpression of FVIII protein comprises a regulatory switch that serves tofine tune expression of the FVIII protein. For example, it can serve asa biocontainment function of the ceDNA vector. In some embodiments, theswitch is an “ON/OFF” switch that is designed to start or stop (i.e.,shut down) expression of FVIII protein in the ceDNA vector in acontrollable and regulatable fashion. In some embodiments, the switchcan include a “kill switch” that can instruct the cell comprising theceDNA vector to undergo cell programmed death once the switch isactivated. Exemplary regulatory switches encompassed for use in a ceDNAvector for expression of FVIII protein can be used to regulate theexpression of a transgene, and are more fully discussed in Internationalapplication PCT/US18/49996, which is incorporated herein in its entiretyby reference

(i) Binary Regulatory Switches

In some embodiments, the ceDNA vector for expression of FVIII proteincomprises a regulatory switch that can serve to controllably modulateexpression of FVIII protein. For example, the expression cassettelocated between the ITRs of the ceDNA vector may additionally comprise aregulatory region, e.g., a promoter, cis-element, repressor, enhanceretc., that is operatively linked to the nucleic acid sequence encodingFVIII protein, where the regulatory region is regulated by one or morecofactors or exogenous agents. By way of example only, regulatoryregions can be modulated by small molecule switches or inducible orrepressible promoters. Non-limiting examples of inducible promoters arehormone-inducible or metal-inducible promoters. Other exemplaryinducible promoters/enhancer elements include, but are not limited to,an RU486-inducible promoter, an ecdysone-inducible promoter, arapamycin-inducible promoter, and a metallothionein promoter.

(ii) Small molecule Regulatory Switches

A variety of art-known small-molecule based regulatory switches areknown in the art and can be combined with the ceDNA vectors forexpression of FVIII protein as disclosed herein to form aregulatory-switch controlled ceDNA vector. In some embodiments, theregulatory switch can be selected from any one or a combination of: anorthogonal ligand/nuclear receptor pair, for example retinoid receptorvariant/LG335 and GRQCIMFI, along with an artificial promotercontrolling expression of the operatively linked transgene, such as thatas disclosed in Taylor, et al. BMC Biotechnology 10 (2010): 15;engineered steroid receptors, e.g., modified progesterone receptor witha C-terminal truncation that cannot bind progesterone but binds RU486(mifepristone) (U.S. Pat. No. 5,364,791); an ecdysone receptor fromDrosophila and their ecdysteroid ligands (Saez, et al., PNAS,97(26)(2000), 14512-14517; or a switch controlled by the antibiotictrimethoprim (TMP), as disclosed in Sando R 3^(rd); Nat Methods. 2013,10(11):1085-8. In some embodiments, the regulatory switch to control thetransgene or expressed by the ceDNA vector is a pro-drug activationswitch, such as that disclosed in U.S. Pat. Nos. 8,771,679, and6,339,070.

(iii) “Passcode” Regulatory Switches

In some embodiments the regulatory switch can be a “passcode switch” or“passcode circuit”. Passcode switches allow fine tuning of the controlof the expression of the transgene from the ceDNA vector when specificconditions occur—that is, a combination of conditions need to be presentfor transgene expression and/or repression to occur. For example, forexpression of a transgene to occur at least conditions A and B mustoccur. A passcode regulatory switch can be any number of conditions,e.g., at least 2, or at least 3, or at least 4, or at least 5, or atleast 6 or at least 7 or more conditions to be present for transgeneexpression to occur. In some embodiments, at least 2 conditions (e.g.,A, B conditions) need to occur, and in some embodiments, at least 3conditions need to occur (e.g., A, B and C, or A, B and D). By way of anexample only, for gene expression from a ceDNA to occur that has apasscode “ABC” regulatory switch, conditions A, B and C must be present.Conditions A, B and C could be as follows; condition A is the presenceof a condition or disease, condition B is a hormonal response, andcondition C is a response to the transgene expression. For example, ifthe transgene edits a defective EPO gene, Condition A is the presence ofChronic Kidney Disease (CKD), Condition B occurs if the subject hashypoxic conditions in the kidney, Condition C is thatErythropoietin-producing cells (EPC) recruitment in the kidney isimpaired; or alternatively, HIF-2 activation is impaired. Once theoxygen levels increase or the desired level of EPO is reached, thetransgene turns off again until 3 conditions occur, turning it back on.

In some embodiments, a passcode regulatory switch or “Passcode circuit”encompassed for use in the ceDNA vector comprises hybrid transcriptionfactors (TFs) to expand the range and complexity of environmentalsignals used to define biocontainment conditions. As opposed to adeadman switch which triggers cell death in the presence of apredetermined condition, the “passcode circuit” allows cell survival ortransgene expression in the presence of a particular “passcode”, and canbe easily reprogrammed to allow transgene expression and/or cellsurvival only when the predetermined environmental condition or passcodeis present.

Any and all combinations of regulatory switches disclosed herein, e.g.,small molecule switches, nucleic acid-based switches, smallmolecule-nucleic acid hybrid switches, post-transcriptional transgeneregulation switches, post-translational regulation, radiation-controlledswitches, hypoxia-mediated switches and other regulatory switches knownby persons of ordinary skill in the art as disclosed herein can be usedin a passcode regulatory switch as disclosed herein. Regulatory switchesencompassed for use are also discussed in the review article Kis et al.,J R Soc Interface. 12: 20141000 (2015), and summarized in Table 1 ofKis. In some embodiments, a regulatory switch for use in a passcodesystem can be selected from any or a combination of the switchesdisclosed in Table 11 of International Patent ApplicationPCT/US18/49996, which is incorporated herein in its entirety byreference.

(iv) Nucleic Acid-Based Regulatory Switches to Control TransgeneExpression

In some embodiments, the regulatory switch to control the expression ofFVIII protein by the ceDNA is based on a nucleic acid based controlmechanism. Exemplary nucleic acid control mechanisms are known in theart and are envisioned for use. For example, such mechanisms includeriboswitches, such as those disclosed in, e.g., US2009/0305253,US2008/0269258, US2017/0204477, WO2018026762A1, U.S. Pat. No. 9,222,093and EP application EP288071, and disclosed in the review by Villa J K etal., Microbiol Spectr. 2018 May; 6(3). Also included aremetabolite-responsive transcription biosensors, such as those disclosedin WO2018/075486 and WO2017/147585. Other art-known mechanismsenvisioned for use include silencing of the transgene with an siRNA orRNAi molecule (e.g., miR, shRNA). For example, the ceDNA vector cancomprise a regulatory switch that encodes a RNAi molecule that iscomplementary to the two part of the transgene expressed by the ceDNAvector. When such RNAi is expressed even if the transgene (e.g., FVIIIprotein) is expressed by the ceDNA vector, it will be silenced by thecomplementary RNAi molecule, and when the RNAi is not expressed when thetransgene is expressed by the ceDNA vector the transgene (e.g., FVIIIprotein) is not silenced by the RNAi.

In some embodiments, the regulatory switch is a tissue-specificself-inactivating regulatory switch, for example as disclosed inUS2002/0022018, whereby the regulatory switch deliberately switchestransgene (e.g., FVIII protein) off at a site where transgene expressionmight otherwise be disadvantageous. In some embodiments, the regulatoryswitch is a recombinase reversible gene expression system, for exampleas disclosed in US2014/0127162 and U.S. Pat. No. 8,324,436.

(v) Post-Transcriptional and Post-Translational Regulatory Switches.

In some embodiments, the regulatory switch to control the expression ofFVIII protein by the ceDNA vector is a post-transcriptional modificationsystem. For example, such a regulatory switch can be an aptazymeriboswitch that is sensitive to tetracycline or theophylline, asdisclosed in US2018/0119156, GB201107768, WO2001/064956A3, EP Patent2707487 and Beilstein et al., ACS Synth. Biol., 2015, 4 (5), pp 526-534;Zhong et al., Elife. 2016 Nov. 2; 5. pii: e18858. In some embodiments,it is envisioned that a person of ordinary skill in the art could encodeboth the transgene and an inhibitory siRNA which contains a ligandsensitive (OFF-switch) aptamer, the net result being a ligand sensitiveON-switch.

(vi) Other Exemplary Regulatory Switches

Any known regulatory switch can be used in the ceDNA vector to controlthe expression of FVIII protein by the ceDNA vector, including thosetriggered by environmental changes. Additional examples include, but arenot limited to; the BOC method of Suzuki et al., Scientific Reports 8;10051 (2018); genetic code expansion and a non-physiologic amino acid;radiation-controlled or ultra-sound controlled on/off switches (see,e.g., Scott S et al., Gene Ther. 2000 July; 7(13):1121-5; U.S. Pat. Nos.5,612,318; 5,571,797; 5,770,581; 5,817,636; and WO1999/025385A1. In someembodiments, the regulatory switch is controlled by an implantablesystem, e.g., as disclosed in U.S. Pat. No. 7,840,263; US2007/0190028A1where gene expression is controlled by one or more forms of energy,including electromagnetic energy, that activates promoters operativelylinked to the transgene in the ceDNA vector.

In some embodiments, a regulatory switch envisioned for use in the ceDNAvector is a hypoxia-mediated or stress-activated switch, e.g., such asthose disclosed in WO1999060142A2, U.S. Pat. Nos. 5,834,306; 6,218,179;6,709,858; US2015/0322410; Greco et al., (2004) Targeted CancerTherapies 9, 5368, as well as FROG, TOAD and NRSE elements andconditionally inducible silence elements, including hypoxia responseelements (HREs), inflammatory response elements (IREs) and shear-stressactivated elements (SSAEs), e.g., as disclosed in U.S. Pat. No.9,394,526. Such an embodiment is useful for turning on expression of thetransgene from the ceDNA vector after ischemia or in ischemic tissues,and/or tumors.

(vii) Kill Switches

Other embodiments described herein relate to a ceDNA vector forexpression of FVIII protein as described herein comprising a killswitch. A kill switch as disclosed herein enables a cell comprising theceDNA vector to be killed or undergo programmed cell death as a means topermanently remove an introduced ceDNA vector from the subject's system.It will be appreciated by one of ordinary skill in the art that use ofkill switches in the ceDNA vectors for expression of FVIII protein wouldbe typically coupled with targeting of the ceDNA vector to a limitednumber of cells that the subject can acceptably lose or to a cell typewhere apoptosis is desirable (e.g., cancer cells). In all aspects, a“kill switch” as disclosed herein is designed to provide rapid androbust cell killing of the cell comprising the ceDNA vector in theabsence of an input survival signal or other specified condition. Statedanother way, a kill switch encoded by a ceDNA vector for expression ofFVIII protein as described herein can restrict cell survival of a cellcomprising a ceDNA vector to an environment defined by specific inputsignals. Such kill switches serve as a biological biocontainmentfunction should it be desirable to remove the ceDNA vector e expressionof FVIII protein in a subject or to ensure that it will not express theencoded FVIII protein.

Other kill switches known to a person of ordinary skill in the art areencompassed for use in the ceDNA vector for expression of FVIII proteinas disclosed herein, e.g., as disclosed in 052010/0175141;052013/0009799; 052011/0172826; 052013/0109568, as well as kill switchesdisclosed in Jusiak et al., Reviews in Cell Biology and molecularMedicine; 2014; 1-56; Kobayashi et al., PNAS, 2004; 101; 8419-9;Marchisio et al., Int. Journal of Biochem and Cell Biol., 2011; 43;310-319; and in Reinshagen et al., Science Translational Medicine, 2018,11.

Accordingly, in some embodiments, the ceDNA vector for expression ofFVIII protein can comprise a kill switch nucleic acid construct, whichcomprises the nucleic acid encoding an effector toxin or reporterprotein, where the expression of the effector toxin (e.g., a deathprotein) or reporter protein is controlled by a predetermined condition.For example, a predetermined condition can be the presence of anenvironmental agent, such as, e.g., an exogenous agent, without whichthe cell will default to expression of the effector toxin (e.g., a deathprotein) and be killed. In alternative embodiments, a predeterminedcondition is the presence of two or more environmental agents, e.g., thecell will only survive when two or more necessary exogenous agents aresupplied, and without either of which, the cell comprising the ceDNAvector is killed.

In some embodiments, the ceDNA vector for expression of FVIII protein ismodified to incorporate a kill-switch to destroy the cells comprisingthe ceDNA vector to effectively terminate the in vivo expression of thetransgene being expressed by the ceDNA vector (e.g., expression of FVIIIprotein). Specifically, the ceDNA vector is further geneticallyengineered to express a switch-protein that is not functional inmammalian cells under normal physiological conditions. Only uponadministration of a drug or environmental condition that specificallytargets this switch-protein, the cells expressing the switch-proteinwill be destroyed thereby terminating the expression of the therapeuticprotein or peptide. For instance, it was reported that cells expressingHSV-thymidine kinase can be killed upon administration of drugs, such asganciclovir and cytosine deaminase. See, for example, Dey and Evans,Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase(HSV-TK), in Targets in Gene Therapy, edited by You (2011); andBeltinger et al., Proc. Natl. Acad. Sci. USA 96(15):8699-8704 (1999). Insome embodiments the ceDNA vector can comprise a siRNA kill switchreferred to as DISE (Death Induced by Survival gene Elimination)(Murmann et al., Oncotarget. 2017; 8:84643-84658. Induction of DISE inovarian cancer cells in vivo).

VI. Detailed Method of Production of a ceDNA Vector A. Production inGeneral

Certain methods for the production of a ceDNA vector for expression ofFVIII protein comprising an asymmetrical ITR pair or symmetrical ITRpair as defined herein is described in section IV of Internationalapplication PCT/US18/49996 filed Sep. 7, 2018, which is incorporatedherein in its entirety by reference. In some embodiments, a ceDNA vectorfor expression of FVIII protein as disclosed herein can be producedusing insect cells, as described herein. In alternative embodiments, aceDNA vector for expression of FVIII protein as disclosed herein can beproduced synthetically and in some embodiments, in a cell-free method,as disclosed on International Application PCT/US19/14122, filed Jan. 18,2019, which is incorporated herein in its entirety by reference.

As described herein, in one embodiment, a ceDNA vector for expression ofFVIII protein can be obtained, for example, by the process comprisingthe steps of: a) incubating a population of host cells (e.g. insectcells) harboring the polynucleotide expression construct template (e.g.,a ceDNA-plasmid, a ceDNA-Bacmid, and/or a ceDNA-baculovirus), which isdevoid of viral capsid coding sequences, in the presence of a Repprotein under conditions effective and for a time sufficient to induceproduction of the ceDNA vector within the host cells, and wherein thehost cells do not comprise viral capsid coding sequences; and b)harvesting and isolating the ceDNA vector from the host cells. Thepresence of Rep protein induces replication of the vector polynucleotidewith a modified ITR to produce the ceDNA vector in a host cell. However,no viral particles (e.g. AAV virions) are expressed. Thus, there is nosize limitation such as that naturally imposed in AAV or otherviral-based vectors.

The presence of the ceDNA vector isolated from the host cells can beconfirmed by digesting DNA isolated from the host cell with arestriction enzyme having a single recognition site on the ceDNA vectorand analyzing the digested DNA material on a non-denaturing gel toconfirm the presence of characteristic bands of linear and continuousDNA as compared to linear and non-continuous DNA.

In yet another aspect, the invention provides for use of host cell linesthat have stably integrated the DNA vector polynucleotide expressiontemplate (ceDNA template) into their own genome in production of thenon-viral DNA vector, e.g. as described in Lee, L. et al. (2013) PlosOne 8(8): e69879. Preferably, Rep is added to host cells at an MOI ofabout 3. When the host cell line is a mammalian cell line, e.g., HEK293cells, the cell lines can have polynucleotide vector template stablyintegrated, and a second vector such as herpes virus can be used tointroduce Rep protein into cells, allowing for the excision andamplification of ceDNA in the presence of Rep and helper virus.

In one embodiment, the host cells used to make the ceDNA vectors forexpression of FVIII protein as described herein are insect cells, andbaculovirus is used to deliver both the polynucleotide that encodes Repprotein and the non-viral DNA vector polynucleotide expression constructtemplate for ceDNA, e.g., as described in FIGS. 4A-4C and Example 1. Insome embodiments, the host cell is engineered to express Rep protein.

The ceDNA vector is then harvested and isolated from the host cells. Thetime for harvesting and collecting ceDNA vectors described herein fromthe cells can be selected and optimized to achieve a high-yieldproduction of the ceDNA vectors. For example, the harvest time can beselected in view of cell viability, cell morphology, cell growth, etc.In one embodiment, cells are grown under sufficient conditions andharvested a sufficient time after baculoviral infection to produce ceDNAvectors but before a majority of cells start to die because of thebaculoviral toxicity. The DNA vectors can be isolated using plasmidpurification kits such as Qiagen Endo-Free Plasmid kits. Other methodsdeveloped for plasmid isolation can be also adapted for DNA vectors.Generally, any nucleic acid purification methods can be adopted.

The DNA vectors can be purified by any means known to those of skill inthe art for purification of DNA. In one embodiment, ceDNA vectors arepurified as DNA molecules. In another embodiment, the ceDNA vectors arepurified as exosomes or microparticles.

The presence of the ceDNA vector for expression of FVIII protein can beconfirmed by digesting the vector DNA isolated from the cells with arestriction enzyme having a single recognition site on the DNA vectorand analyzing both digested and undigested DNA material using gelelectrophoresis to confirm the presence of characteristic bands oflinear and continuous DNA as compared to linear and non-continuous DNA.FIG. 4C and FIG. 4D illustrate one embodiment for identifying thepresence of the closed ended ceDNA vectors produced by the processesherein.

B. ceDNA Plasmid

A ceDNA-plasmid is a plasmid used for later production of a ceDNA vectorfor expression of FVIII protein. In some embodiments, a ceDNA-plasmidcan be constructed using known techniques to provide at least thefollowing as operatively linked components in the direction oftranscription: (1) a modified 5′ ITR sequence; (2) an expressioncassette containing a cis-regulatory element, for example, a promoter,inducible promoter, regulatory switch, enhancers and the like; and (3) amodified 3′ ITR sequence, where the 3′ ITR sequence is symmetricrelative to the 5′ ITR sequence. In some embodiments, the expressioncassette flanked by the ITRs comprises a cloning site for introducing anexogenous sequence. The expression cassette replaces the rep and capcoding regions of the AAV genomes.

In one aspect, a ceDNA vector for expression of FVIII protein isobtained from a plasmid, referred to herein as a “ceDNA-plasmid”encoding in this order: a first adeno-associated virus (AAV) invertedterminal repeat (ITR), an expression cassette comprising a transgene,and a mutated or modified AAV ITR, wherein said ceDNA-plasmid is devoidof AAV capsid protein coding sequences. In alternative embodiments, theceDNA-plasmid encodes in this order: a first (or 5′) modified or mutatedAAV ITR, an expression cassette comprising a transgene, and a second (or3′) modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsidprotein coding sequences, and wherein the 5′ and 3′ ITRs are symmetricrelative to each other. In alternative embodiments, the ceDNA-plasmidencodes in this order: a first (or 5′) modified or mutated AAV ITR, anexpression cassette comprising a transgene, and a second (or 3′) mutatedor modified AAV ITR, wherein said ceDNA-plasmid is devoid of AAV capsidprotein coding sequences, and wherein the 5′ and 3′ modified ITRs arehave the same modifications (i.e., they are inverse complement orsymmetric relative to each other).

In a further embodiment, the ceDNA-plasmid system is devoid of viralcapsid protein coding sequences (i.e. it is devoid of AAV capsid genesbut also of capsid genes of other viruses). In addition, in a particularembodiment, the ceDNA-plasmid is also devoid of AAV Rep protein codingsequences. Accordingly, in a preferred embodiment, ceDNA-plasmid isdevoid of functional AAV cap and AAV rep genes GG-3′ for AAV2) plus avariable palindromic sequence allowing for hairpin formation.

A ceDNA-plasmid of the present invention can be generated using naturalnucleotide sequences of the genomes of any AAV serotypes well known inthe art. In one embodiment, the ceDNA-plasmid backbone is derived fromthe AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome. E.g., NCBI: NC002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261;Kotin and Smith, The Springer Index of Viruses, available at the URLmaintained by Springer (at www web address:oesys.springer.de/viruses/database/mkchapter.asp?virID=42.04.)(note-references to a URL or database refer to the contents of the URL ordatabase as of the effective filing date of this application) In aparticular embodiment, the ceDNA-plasmid backbone is derived from theAAV2 genome. In another particular embodiment, the ceDNA-plasmidbackbone is a synthetic backbone genetically engineered to include atits 5′ and 3′ ITRs derived from one of these AAV genomes.

A ceDNA-plasmid can optionally include a selectable or selection markerfor use in the establishment of a ceDNA vector-producing cell line. Inone embodiment, the selection marker can be inserted downstream (i.e.,3′) of the 3′ ITR sequence. In another embodiment, the selection markercan be inserted upstream (i.e., 5′) of the 5′ ITR sequence. Appropriateselection markers include, for example, those that confer drugresistance. Selection markers can be, for example, a blasticidinS-resistance gene, kanamycin, geneticin, and the like. In a preferredembodiment, the drug selection marker is a blasticidin S-resistancegene.

An exemplary ceDNA (e.g., rAAV0) vector for expression of FVIII proteinis produced from an rAAV plasmid. A method for the production of a rAAVvector, can comprise: (a) providing a host cell with a rAAV plasmid asdescribed above, wherein both the host cell and the plasmid are devoidof capsid protein encoding genes, (b) culturing the host cell underconditions allowing production of an ceDNA genome, and (c) harvestingthe cells and isolating the AAV genome produced from said cells.

C. Exemplary Method of Making the ceDNA Vectors from ceDNA Plasmids

Methods for making capsid-less ceDNA vectors for expression of FVIIIprotein are also provided herein, notably a method with a sufficientlyhigh yield to provide sufficient vector for in vivo experiments.

In some embodiments, a method for the production of a ceDNA vector forexpression of FVIII protein comprises the steps of: (1) introducing thenucleic acid construct comprising an expression cassette and twosymmetric ITR sequences into a host cell (e.g., Sf9 cells), (2)optionally, establishing a clonal cell line, for example, by using aselection marker present on the plasmid, (3) introducing a Rep codinggene (either by transfection or infection with a baculovirus carryingsaid gene) into said insect cell, and (4) harvesting the cell andpurifying the ceDNA vector. The nucleic acid construct comprising anexpression cassette and two ITR sequences described above for theproduction of ceDNA vector can be in the form of a ceDNA plasmid, orBacmid or Baculovirus generated with the ceDNA plasmid as describedbelow. The nucleic acid construct can be introduced into a host cell bytransfection, viral transduction, stable integration, or other methodsknown in the art.

D. Cell Lines

Host cell lines used in the production of a ceDNA vector for expressionof FVIII protein can include insect cell lines derived from Spodopterafrugiperda, such as Sf9 Sf21, or Trichoplusia ni cell, or otherinvertebrate, vertebrate, or other eukaryotic cell lines includingmammalian cells. Other cell lines known to an ordinarily skilled artisancan also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO,COS, MeWo, NIH3T3, A549, HT1 180, monocytes, and mature and immaturedendritic cells. Host cell lines can be transfected for stableexpression of the ceDNA-plasmid for high yield ceDNA vector production.

CeDNA-plasmids can be introduced into Sf9 cells by transienttransfection using reagents (e.g., liposomal, calcium phosphate) orphysical means (e.g., electroporation) known in the art. Alternatively,stable Sf9 cell lines which have stably integrated the ceDNA-plasmidinto their genomes can be established. Such stable cell lines can beestablished by incorporating a selection marker into the ceDNA-plasmidas described above. If the ceDNA-plasmid used to transfect the cell lineincludes a selection marker, such as an antibiotic, cells that have beentransfected with the ceDNA-plasmid and integrated the ceDNA-plasmid DNAinto their genome can be selected for by addition of the antibiotic tothe cell growth media. Resistant clones of the cells can then beisolated by single-cell dilution or colony transfer techniques andpropagated.

E. Isolating and Purifying ceDNA Vectors:

Examples of the process for obtaining and isolating ceDNA vectors aredescribed in FIGS. 4A-4E and the specific examples below. ceDNA-vectorsfor expression of FVIII protein disclosed herein can be obtained from aproducer cell expressing AAV Rep protein(s), further transformed with aceDNA-plasmid, ceDNA-bacmid, or ceDNA-baculovirus. Plasmids useful forthe production of ceDNA vectors include plasmids that encode FVIIIprotein, or plasmids encoding one or more REP proteins.

In one aspect, a polynucleotide encodes the AAV Rep protein (Rep 78 or68) delivered to a producer cell in a plasmid (Rep-plasmid), a bacmid(Rep-bacmid), or a baculovirus (Rep-baculovirus). The Rep-plasmid,Rep-bacmid, and Rep-baculovirus can be generated by methods describedabove.

Methods to produce a ceDNA vector for expression of FVIII protein aredescribed herein. Expression constructs used for generating a ceDNAvector for expression of FVIII protein as described herein can be aplasmid (e.g., ceDNA-plasmids), a Bacmid (e.g., ceDNA-bacmid), and/or abaculovirus (e.g., ceDNA-baculovirus). By way of an example only, aceDNA-vector can be generated from the cells co-infected withceDNA-baculovirus and Rep-baculovirus. Rep proteins produced from theRep-baculovirus can replicate the ceDNA-baculovirus to generateceDNA-vectors. Alternatively, ceDNA vectors for expression of FVIIIprotein can be generated from the cells stably transfected with aconstruct comprising a sequence encoding the AAV Rep protein (Rep78/52)delivered in Rep-plasmids, Rep-bacmids, or Rep-baculovirus.CeDNA-Baculovirus can be transiently transfected to the cells, bereplicated by Rep protein and produce ceDNA vectors.

The bacmid (e.g., ceDNA-bacmid) can be transfected into permissiveinsect cells such as Sf9, Sf21, Tni (Trichoplusia ni) cell, High Fivecell, and generate ceDNA-baculovirus, which is a recombinant baculovirusincluding the sequences comprising the symmetric ITRs and the expressioncassette. ceDNA-baculovirus can be again infected into the insect cellsto obtain a next generation of the recombinant baculovirus. Optionally,the step can be repeated once or multiple times to produce therecombinant baculovirus in a larger quantity.

The time for harvesting and collecting ceDNA vectors for expression ofFVIII protein as described herein from the cells can be selected andoptimized to achieve a high-yield production of the ceDNA vectors. Forexample, the harvest time can be selected in view of cell viability,cell morphology, cell growth, etc. Usually, cells can be harvested aftersufficient time after baculoviral infection to produce ceDNA vectors(e.g., ceDNA vectors) but before majority of cells start to die becauseof the viral toxicity. The ceDNA-vectors can be isolated from the Sf9cells using plasmid purification kits such as Qiagen ENDO-FREE PLASMID®kits. Other methods developed for plasmid isolation can be also adaptedfor ceDNA vectors. Generally, any art-known nucleic acid purificationmethods can be adopted, as well as commercially available DNA extractionkits.

Alternatively, purification can be implemented by subjecting a cellpellet to an alkaline lysis process, centrifuging the resulting lysateand performing chromatographic separation. As one non-limiting example,the process can be performed by loading the supernatant on an ionexchange column (e.g. SARTOBIND Q®) which retains nucleic acids, andthen eluting (e.g. with a 1.2 M NaCl solution) and performing a furtherchromatographic purification on a gel filtration column (e.g. 6 fastflow GE). The capsid-free AAV vector is then recovered by, e.g.,precipitation.

In some embodiments, ceDNA vectors for expression of FVIII protein canalso be purified in the form of exosomes, or microparticles. It is knownin the art that many cell types release not only soluble proteins, butalso complex protein/nucleic acid cargoes via membrane microvesicleshedding (Cocucci et al, 2009; EP 10306226.1) Such vesicles includemicrovesicles (also referred to as microparticles) and exosomes (alsoreferred to as nanovesicles), both of which comprise proteins and RNA ascargo. Microvesicles are generated from the direct budding of the plasmamembrane, and exosomes are released into the extracellular environmentupon fusion of multivesicular endosomes with the plasma membrane. Thus,ceDNA vector-containing microvesicles and/or exosomes can be isolatedfrom cells that have been transduced with the ceDNA-plasmid or a bacmidor baculovirus generated with the ceDNA-plasmid.

Microvesicles can be isolated by subjecting culture medium to filtrationor ultracentrifugation at 20,000×g, and exosomes at 100,000×g. Theoptimal duration of ultracentrifugation can be experimentally-determinedand will depend on the particular cell type from which the vesicles areisolated. Preferably, the culture medium is first cleared by low-speedcentrifugation (e.g., at 2000×g for 5-20 minutes) and subjected to spinconcentration using, e.g., an AMICON® spin column (Millipore, Watford,UK). Microvesicles and exosomes can be further purified via FACS or MACSby using specific antibodies that recognize specific surface antigenspresent on the microvesicles and exosomes. Other microvesicle andexosome purification methods include, but are not limited to,immunoprecipitation, affinity chromatography, filtration, and magneticbeads coated with specific antibodies or aptamers. Upon purification,vesicles are washed with, e.g., phosphate-buffered saline. One advantageof using microvesicles or exosome to deliver ceDNA-containing vesiclesis that these vesicles can be targeted to various cell types byincluding on their membrane proteins recognized by specific receptors onthe respective cell types. (See also EP 10306226)

Another aspect of the invention herein relates to methods of purifyingceDNA vectors from host cell lines that have stably integrated a ceDNAconstruct into their own genome. In one embodiment, ceDNA vectors arepurified as DNA molecules. In another embodiment, the ceDNA vectors arepurified as exosomes or microparticles.

FIG. 5 of International application PCT/US18/49996 shows a gelconfirming the production of ceDNA from multiple ceDNA-plasmidconstructs using the method described in the Examples. The ceDNA isconfirmed by a characteristic band pattern in the gel, as discussed withrespect to FIG. 4D in the Examples.

VII. Pharmaceutical Compositions

In another aspect, pharmaceutical compositions are provided. Thepharmaceutical composition comprises a ceDNA vector for expression ofFVIII protein as described herein and a pharmaceutically acceptablecarrier or diluent.

The ceDNA vectors for expression of FVIII protein as disclosed hereincan be incorporated into pharmaceutical compositions suitable foradministration to a subject for in vivo delivery to cells, tissues, ororgans of the subject. Typically, the pharmaceutical compositioncomprises a ceDNA-vector as disclosed herein and a pharmaceuticallyacceptable carrier. For example, the ceDNA vectors for expression ofFVIII protein as described herein can be incorporated into apharmaceutical composition suitable for a desired route of therapeuticadministration (e.g., parenteral administration). Passive tissuetransduction via high pressure intravenous or intra-arterial infusion,as well as intracellular injection, such as intranuclear microinjectionor intracytoplasmic injection, are also contemplated. Pharmaceuticalcompositions for therapeutic purposes can be formulated as a solution,microemulsion, dispersion, liposomes, or other ordered structuresuitable to high ceDNA vector concentration. Sterile injectablesolutions can be prepared by incorporating the ceDNA vector compound inthe required amount in an appropriate buffer with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization including a ceDNA vector can be formulated to deliver atransgene in the nucleic acid to the cells of a recipient, resulting inthe therapeutic expression of the transgene or donor sequence therein.The composition can also include a pharmaceutically acceptable carrier.

Pharmaceutically active compositions comprising a ceDNA vector forexpression of FVIII protein can be formulated to deliver a transgene forvarious purposes to the cell, e.g., cells of a subject.

Pharmaceutical compositions for therapeutic purposes typically must besterile and stable under the conditions of manufacture and storage. Thecomposition can be formulated as a solution, microemulsion, dispersion,liposomes, or other ordered structure suitable to high ceDNA vectorconcentration. Sterile injectable solutions can be prepared byincorporating the ceDNA vector compound in the required amount in anappropriate buffer with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization.

A ceDNA vector for expression of FVIII protein as disclosed herein canbe incorporated into a pharmaceutical composition suitable for topical,systemic, intra-amniotic, intrathecal, intracranial, intra-arterial,intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal,intra-tissue (e.g., intramuscular, intracardiac, intrahepatic,intrarenal, intracerebral), intrathecal, intravesical, conjunctival(e.g., extra-orbital, intraorbital, retroorbital, intraretinal,subretinal, choroidal, sub-choroidal, intrastromal, intracameral andintravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal)administration. Passive tissue transduction via high pressureintravenous or intraarterial infusion, as well as intracellularinjection, such as intranuclear microinjection or intracytoplasmicinjection, are also contemplated.

In some aspects, the methods provided herein comprise delivering one ormore ceDNA vectors for expression of FVIII protein as disclosed hereinto a host cell. Also provided herein are cells produced by such methods,and organisms (such as animals, plants, or fungi) comprising or producedfrom such cells. Methods of delivery of nucleic acids can includelipofection, nucleofection, microinjection, biolistics, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagentsare sold commercially (e.g., Transfectam™ and Lipofectin™). Delivery canbe to cells (e.g., in vitro or ex vivo administration) or target tissues(e.g., in vivo administration).

Various techniques and methods are known in the art for deliveringnucleic acids to cells. For example, nucleic acids, such as ceDNA forexpression of FVIII protein can be formulated into lipid nanoparticles(LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, orcore-shell nanoparticles. Typically, LNPs are composed of nucleic acid(e.g., ceDNA) molecules, one or more ionizable or cationic lipids (orsalts thereof), one or more non-ionic or neutral lipids (e.g., aphospholipid), a molecule that prevents aggregation (e.g., PEG or aPEG-lipid conjugate), and optionally a sterol (e.g., cholesterol).

Another method for delivering nucleic acids, such as ceDNA forexpression of FVIII protein to a cell is by conjugating the nucleic acidwith a ligand that is internalized by the cell. For example, the ligandcan bind a receptor on the cell surface and internalized viaendocytosis. The ligand can be covalently linked to a nucleotide in thenucleic acid. Exemplary conjugates for delivering nucleic acids into acell are described, example, in WO2015/006740, WO2014/025805,WO2012/037254, WO2009/082606, WO2009/073809, WO2009/018332,WO2006/112872, WO2004/090108, WO2004/091515 and WO2017/177326.

Nucleic acids, such as ceDNA vectors for expression of FVIII protein canalso be delivered to a cell by transfection. Useful transfection methodsinclude, but are not limited to, lipid-mediated transfection, cationicpolymer-mediated transfection, or calcium phosphate precipitation.Transfection reagents are well known in the art and include, but are notlimited to, TurboFect Transfection Reagent (Thermo Fisher Scientific),Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASS™ P ProteinTransfection Reagent (New England Biolabs), CHARIOT™ Protein DeliveryReagent (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMDMillipore), 293fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (ThermoFisher Scientific), LIPOFECTAMINE™ (Thermo Fisher Scientific),LIPOFECTIN™ (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN™ (ThermoFisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific),LIPOFECTACE™, FUGENE™ (Roche, Basel, Switzerland), FUGENE™ HD (Roche),TRANSFECTAM™ (Transfectam, Promega, Madison, Wis.), TFX-10™ (Promega),TFX-20™ (Promega), TFX-50™ (Promega), TRANSFECTIN™ (BioRad, Hercules,Calif.), SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen, Valencia, Calif.),DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems, SanDiego, Calif.), DHARMAFECT 1™ (Dharmacon, Lafayette, Colo.), DHARMAFECT2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon),ESCORT™ III (Sigma, St. Louis, Mo.), and ESCORT™ IV (Sigma ChemicalCo.). Nucleic acids, such as ceDNA, can also be delivered to a cell viamicrofluidics methods known to those of skill in the art.

ceDNA vectors for expression of FVIII protein as described herein canalso be administered directly to an organism for transduction of cellsin vivo. Administration is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cellsincluding, but not limited to, injection, infusion, topical applicationand electroporation. Suitable methods of administering such nucleicacids are available and well known to those of skill in the art, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of a nucleic acid vector ceDNA vector forexpression of FVIII protein as disclosed herein can be delivered intohematopoietic stem cells, for example, by the methods as described, forexample, in U.S. Pat. No. 5,928,638.

The ceDNA vectors for expression of FVIII protein in accordance with thepresent invention can be added to liposomes for delivery to a cell ortarget organ in a subject. Liposomes are vesicles that possess at leastone lipid bilayer. Liposomes are typical used as carriers fordrug/therapeutic delivery in the context of pharmaceutical development.They work by fusing with a cellular membrane and repositioning its lipidstructure to deliver a drug or active pharmaceutical ingredient (API).Liposome compositions for such delivery are composed of phospholipids,especially compounds having a phosphatidylcholine group, however thesecompositions may also include other lipids. Exemplary liposomes andliposome formulations, including but not limited to polyethylene glycol(PEG)-functional group containing compounds are disclosed inInternational Application PCT/US2018/050042, filed on Sep. 7, 2018 andin International application PCT/US2018/064242, filed on Dec. 6, 2018,e.g., see the section entitled “Pharmaceutical Formulations”.

Various delivery methods known in the art or modification thereof can beused to deliver ceDNA vectors in vitro or in vivo. For example, in someembodiments, ceDNA vectors for expression of FVIII protein are deliveredby making transient penetration in cell membrane by mechanical,electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNAentrance into the targeted cells is facilitated. For example, a ceDNAvector can be delivered by transiently disrupting cell membrane bysqueezing the cell through a size-restricted channel or by other meansknown in the art. In some cases, a ceDNA vector alone is directlyinjected as naked DNA into any one of: any one or more tissues selectedfrom: liver, kidneys, gallbladder, prostate, adrenal gland, heart,intestine, lung, and stomach, skin, thymus, cardiac muscle or skeletalmuscle. In some cases, a ceDNA vector is delivered by gene gun. Gold ortungsten spherical particles (1-3 μm diameter) coated with capsid-freeAAV vectors can be accelerated to high speed by pressurized gas topenetrate into target tissue cells.

Compositions comprising a ceDNA vector for expression of FVIII proteinand a pharmaceutically acceptable carrier are specifically contemplatedherein. In some embodiments, the ceDNA vector is formulated with a lipiddelivery system, for example, liposomes as described herein. In someembodiments, such compositions are administered by any route desired bya skilled practitioner. The compositions may be administered to asubject by different routes including orally, parenterally,sublingually, transdermally, rectally, transmucosally, topically, viainhalation, via buccal administration, intrapleurally, intravenous,intra-arterial, intraperitoneal, subcutaneous, intramuscular, intranasalintrathecal, and intraarticular or combinations thereof. For veterinaryuse, the composition may be administered as a suitably acceptableformulation in accordance with normal veterinary practice. Theveterinarian may readily determine the dosing regimen and route ofadministration that is most appropriate for a particular animal Thecompositions may be administered by traditional syringes, needlelessinjection devices, “microprojectile bombardment gene guns”, or otherphysical methods such as electroporation (“EP”), hydrodynamic methods,or ultrasound.

In some cases, a ceDNA vector for expression of FVIII protein isdelivered by hydrodynamic injection, which is a simple and highlyefficient method for direct intracellular delivery of any water-solublecompounds and particles into internal organs and skeletal muscle in anentire limb.

In some cases, ceDNA vectors for expression of FVIII protein aredelivered by ultrasound by making nanoscopic pores in membrane tofacilitate intracellular delivery of DNA particles into cells ofinternal organs or tumors, so the size and concentration of plasmid DNAhave great role in efficiency of the system. In some cases, ceDNAvectors are delivered by magnetofection by using magnetic fields toconcentrate particles containing nucleic acid into the target cells.

In some cases, chemical delivery systems can be used, for example, byusing nanomeric complexes, which include compaction of negativelycharged nucleic acid by polycationic nanomeric particles, belonging tocationic liposome/micelle or cationic polymers. Cationic lipids used forthe delivery method includes, but not limited to monovalent cationiclipids, polyvalent cationic lipids, guanidine containing compounds,cholesterol derivative compounds, cationic polymers, (e.g.,poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers),and lipid-polymer hybrid.

A. Exosomes

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is delivered by being packaged in an exosome. Exosomesare small membrane vesicles of endocytic origin that are released intothe extracellular environment following fusion of multivesicular bodieswith the plasma membrane. Their surface consists of a lipid bilayer fromthe donor cell's cell membrane, they contain cytosol from the cell thatproduced the exosome, and exhibit membrane proteins from the parentalcell on the surface. Exosomes are produced by various cell typesincluding epithelial cells, B and T lymphocytes, mast cells (MC) as wellas dendritic cells (DC). Some embodiments, exosomes with a diameterbetween 10 nm and 1 μm, between 20 nm and 500 nm, between 30 nm and 250nm, between 50 nm and 100 nm are envisioned for use. Exosomes can beisolated for a delivery to target cells using either their donor cellsor by introducing specific nucleic acids into them. Various approachesknown in the art can be used to produce exosomes containing capsid-freeAAV vectors of the present invention.

B. Microparticle/Nanoparticles

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is delivered by a lipid nanoparticle. Generally, lipidnanoparticles comprise an ionizable amino lipid (e.g.,heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate,DLin-MC3-DMA, a phosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and acoat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), forexample as disclosed by Tam et al. (2013). Advances in LipidNanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.

In some embodiments, a lipid nanoparticle has a mean diameter betweenabout 10 and about 1000 nm. In some embodiments, a lipid nanoparticlehas a diameter that is less than 300 nm. In some embodiments, a lipidnanoparticle has a diameter between about 10 and about 300 nm. In someembodiments, a lipid nanoparticle has a diameter that is less than 200nm. In some embodiments, a lipid nanoparticle has a diameter betweenabout 25 and about 200 nm. In some embodiments, a lipid nanoparticlepreparation (e.g., composition comprising a plurality of lipidnanoparticles) has a size distribution in which the mean size (e.g.,diameter) is about 70 nm to about 200 nm, and more typically the meansize is about 100 nm or less.

Various lipid nanoparticles known in the art can be used to deliverceDNA vector for expression of FVIII protein as disclosed herein. Forexample, various delivery methods using lipid nanoparticles aredescribed in U.S. Pat. Nos. 9,404,127, 9,006,417 and 9,518,272.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is delivered by a gold nanoparticle. Generally, anucleic acid can be covalently bound to a gold nanoparticle ornon-covalently bound to a gold nanoparticle (e.g., bound by acharge-charge interaction), for example as described by Ding et al.(2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 22(6);1075-1083. In some embodiments, gold nanoparticle-nucleic acidconjugates are produced using methods described, for example, in U.S.Pat. No. 6,812,334.

C. Conjugates

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is conjugated (e.g., covalently bound to an agent thatincreases cellular uptake. An “agent that increases cellular uptake” isa molecule that facilitates transport of a nucleic acid across a lipidmembrane. For example, a nucleic acid can be conjugated to a lipophiliccompound (e.g., cholesterol, tocopherol, etc.), a cell penetratingpeptide (CPP) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines(e.g., spermine). Further examples of agents that increase cellularuptake are disclosed, for example, in Winkler (2013). Oligonucleotideconjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is conjugated to a polymer (e.g., a polymeric molecule)or a folate molecule (e.g., folic acid molecule). Generally, delivery ofnucleic acids conjugated to polymers is known in the art, for example asdescribed in WO2000/34343 and WO2008/022309. In some embodiments, aceDNA vector for expression of FVIII protein as disclosed herein isconjugated to a poly(amide) polymer, for example as described by U.S.Pat. No. 8,987,377. In some embodiments, a nucleic acid described by thedisclosure is conjugated to a folic acid molecule as described in U.S.Pat. No. 8,507,455.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is conjugated to a carbohydrate, for example asdescribed in U.S. Pat. No. 8,450,467.

D. Nanocapsule

Alternatively, nanocapsule formulations of a ceDNA vector for expressionof FVIII protein as disclosed herein can be used. Nanocapsules cangenerally entrap substances in a stable and reproducible way. To avoidside effects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for use.

E. Liposomes

The ceDNA vectors for expression of FVIII protein in accordance with thepresent invention can be added to liposomes for delivery to a cell ortarget organ in a subject. Liposomes are vesicles that possess at leastone lipid bilayer. Liposomes are typical used as carriers fordrug/therapeutic delivery in the context of pharmaceutical development.They work by fusing with a cellular membrane and repositioning its lipidstructure to deliver a drug or active pharmaceutical ingredient (API).Liposome compositions for such delivery are composed of phospholipids,especially compounds having a phosphatidylcholine group, however thesecompositions may also include other lipids.

The formation and use of liposomes are generally known to those of skillin the art. Liposomes have been developed with improved serum stabilityand circulation half-times (U.S. Pat. No. 5,741,516). Further, variousmethods of liposome and liposome like preparations as potential drugcarriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157;5,565,213; 5,738,868 and 5,795,587).

F. Exemplary Liposome and Lipid Nanoparticle (LNP) Compositions

The ceDNA vectors for expression of FVIII protein in accordance with thepresent invention can be added to liposomes for delivery to a cell,e.g., a cell in need of expression of the transgene. Liposomes arevesicles that possess at least one lipid bilayer. Liposomes are typicalused as carriers for drug/therapeutic delivery in the context ofpharmaceutical development. They work by fusing with a cellular membraneand repositioning its lipid structure to deliver a drug or activepharmaceutical ingredient (API). Liposome compositions for such deliveryare composed of phospholipids, especially compounds having aphosphatidylcholine group, however these compositions may also includeother lipids.

Lipid nanoparticles (LNPs) comprising ceDNA vectors are disclosed inInternational Application PCT/US2018/050042, filed on Sep. 7, 2018, andInternational Application PCT/US2018/064242, filed on Dec. 6, 2018 whichare incorporated herein in their entirety and envisioned for use in themethods and compositions for ceDNA vectors for expression of FVIIIprotein as disclosed herein.

In some aspects, the disclosure provides for a liposome formulation thatincludes one or more compounds with a polyethylene glycol (PEG)functional group (so-called “PEG-ylated compounds”) which can reduce theimmunogenicity/antigenicity of, provide hydrophilicity andhydrophobicity to the compound(s) and reduce dosage frequency. Or theliposome formulation simply includes polyethylene glycol (PEG) polymeras an additional component. In such aspects, the molecular weight of thePEG or PEG functional group can be from 62 Da to about 5,000 Da.

In some aspects, the disclosure provides for a liposome formulation thatwill deliver an API with extended release or controlled release profileover a period of hours to weeks. In some related aspects, the liposomeformulation may comprise aqueous chambers that are bound by lipidbilayers. In other related aspects, the liposome formulationencapsulates an API with components that undergo a physical transitionat elevated temperature which releases the API over a period of hours toweeks.

In some aspects, the liposome formulation comprises sphingomyelin andone or more lipids disclosed herein. In some aspects, the liposomeformulation comprises optisomes.

In some aspects, the disclosure provides for a liposome formulation thatincludes one or more lipids selected from:N-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt,(distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxy polyethyleneglycol)-conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine);PEG (polyethylene glycol); DSPE(distearoyl-sn-glycero-phosphoethanolamine); DSPC(distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine);DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine);DOPS (dioleoylphosphatidylserine); POPC(palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxypolyethylene glycol); DMPC (dimyristoyl phosphatidylcholine); DMPG(dimyristoyl phosphatidylglycerol); DSPG(distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine);DOPE (dioleoly-sn-glycero-phophoethanolamine); cholesteryl sulphate(CS); dipalmitoylphosphatidylglycerol (DPPG); DOPC(dioleoly-sn-glycero-phosphatidylcholine) or any combination thereof.

In some aspects, the disclosure provides for a liposome formulationcomprising phospholipid, cholesterol and a PEG-ylated lipid in a molarratio of 56:38:5. In some aspects, the liposome formulation's overalllipid content is from 2-16 mg/mL. In some aspects, the disclosureprovides for a liposome formulation comprising a lipid containing aphosphatidylcholine functional group, a lipid containing an ethanolaminefunctional group and a PEG-ylated lipid. In some aspects, the disclosureprovides for a liposome formulation comprising a lipid containing aphosphatidylcholine functional group, a lipid containing an ethanolaminefunctional group and a PEG-ylated lipid in a molar ratio of 3:0.015:2respectively. In some aspects, the disclosure provides for a liposomeformulation comprising a lipid containing a phosphatidylcholinefunctional group, cholesterol and a PEG-ylated lipid. In some aspects,the disclosure provides for a liposome formulation comprising a lipidcontaining a phosphatidylcholine functional group and cholesterol. Insome aspects, the PEG-ylated lipid is PEG-2000-DSPE. In some aspects,the disclosure provides for a liposome formulation comprising DPPG, soyPC, MPEG-DSPE lipid conjugate and cholesterol.

In some aspects, the disclosure provides for a liposome formulationcomprising one or more lipids containing a phosphatidylcholinefunctional group and one or more lipids containing an ethanolaminefunctional group. In some aspects, the disclosure provides for aliposome formulation comprising one or more: lipids containing aphosphatidylcholine functional group, lipids containing an ethanolaminefunctional group, and sterols, e.g. cholesterol. In some aspects, theliposome formulation comprises DOPC/DEPC; and DOPE.

In some aspects, the disclosure provides for a liposome formulationfurther comprising one or more pharmaceutical excipients, e.g. sucroseand/or glycine.

In some aspects, the disclosure provides for a liposome formulation thatis either unilamellar or multilamellar in structure. In some aspects,the disclosure provides for a liposome formulation that comprisesmulti-vesicular particles and/or foam-based particles. In some aspects,the disclosure provides for a liposome formulation that are larger inrelative size to common nanoparticles and about 150 to 250 nm in size.In some aspects, the liposome formulation is a lyophilized powder.

In some aspects, the disclosure provides for a liposome formulation thatis made and loaded with ceDNA vectors disclosed or described herein, byadding a weak base to a mixture having the isolated ceDNA outside theliposome. This addition increases the pH outside the liposomes toapproximately 7.3 and drives the API into the liposome. In some aspects,the disclosure provides for a liposome formulation having a pH that isacidic on the inside of the liposome. In such cases the inside of theliposome can be at pH 4-6.9, and more preferably pH 6.5. In otheraspects, the disclosure provides for a liposome formulation made byusing intra-liposomal drug stabilization technology. In such cases,polymeric or non-polymeric highly charged anions and intra-liposomaltrapping agents are utilized, e.g. polyphosphate or sucrose octasulfate.

In some aspects, the disclosure provides for a lipid nanoparticlecomprising ceDNA and an ionizable lipid. For example, a lipidnanoparticle formulation that is made and loaded with ceDNA obtained bythe process as disclosed in International Application PCT/US2018/050042,filed on Sep. 7, 2018, which is incorporated herein. This can beaccomplished by high energy mixing of ethanolic lipids with aqueousceDNA at low pH which protonates the ionizable lipid and providesfavorable energetics for ceDNA/lipid association and nucleation ofparticles. The particles can be further stabilized through aqueousdilution and removal of the organic solvent. The particles can beconcentrated to the desired level.

Generally, the lipid particles are prepared at a total lipid to ceDNA(mass or weight) ratio of from about 10:1 to 30:1. In some embodiments,the lipid to ceDNA ratio (mass/mass ratio; w/w ratio) can be in therange of from about 1:1 to about 25:1, from about 10:1 to about 14:1,from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids andceDNA can be adjusted to provide a desired N/P ratio, for example, N/Pratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipidparticle formulation's overall lipid content can range from about 5mg/ml to about 30 mg/mL.

The ionizable lipid is typically employed to condense the nucleic acidcargo, e.g., ceDNA at low pH and to drive membrane association andfusogenicity. Generally, ionizable lipids are lipids comprising at leastone amino group that is positively charged or becomes protonated underacidic conditions, for example at pH of 6.5 or lower. Ionizable lipidsare also referred to as cationic lipids herein.

Exemplary ionizable lipids are described in International PCT patentpublications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245,WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085,WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528,WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120,WO2012/044638, WO2012/054365, WO2011/090965, WO2013/016058,WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740,WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373,WO2011/071860, WO2009/132131, WO2010/048536, WO2010/088537,WO2010/054401, WO2010/054406, WO2010/054405, WO2010/054384,WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106,WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126803,WO2006/007712, WO2011/038160, WO2005/121348, WO2011/066651,WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043,WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823,WO2015/095346, and WO2013/086354, and US patent publications052016/0311759, 052015/0376115, 052016/0151284, 052017/0210697,052015/0140070, 052013/0178541, 052013/0303587, 052015/0141678,052015/0239926, 052016/0376224, 052017/0119904, 052012/0149894,052015/0057373, 052013/0090372, 052013/0274523, US2013/0274504,US2013/0274504, 052009/0023673, US2012/0128760, US2010/0324120,052014/0200257, 052015/0203446, 052018/0005363, 052014/0308304,052013/0338210, 052012/0101148, 052012/0027796, 052012/0058144,052013/0323269, 052011/0117125, 052011/0256175, 052012/0202871,052011/0076335, 052006/0083780, 052013/0123338, 052015/0064242,052006/0051405, US2013/0065939, 052006/0008910, 052003/0022649,052010/0130588, 052013/0116307, 052010/0062967, 052013/0202684,052014/0141070, 052014/0255472, 052014/0039032, 052018/0028664,052016/0317458, and 052013/0195920, the contents of all of which areincorporated herein by reference in their entirety.

In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) having the following structure:

The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem.Int. Ed Engl. (2012), 51(34): 8529-8533, content of which isincorporated herein by reference in its entirety.

In some embodiments, the ionizable lipid is the lipid ATX-002 asdescribed in WO2015/074085, content of which is incorporated herein byreference in its entirety.

In some embodiments, the ionizable lipid is(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32),as described in WO2012/040184, content of which is incorporated hereinby reference in its entirety.

In some embodiments, the ionizable lipid is Compound 6 or Compound 22 asdescribed in WO2015/199952, content of which is incorporated herein byreference in its entirety.

Without limitations, ionizable lipid can comprise 20-90% (mol) of thetotal lipid present in the lipid nanoparticle. For example, ionizablelipid molar content can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) ofthe total lipid present in the lipid nanoparticle. In some embodiments,ionizable lipid comprises from about 50 mol % to about 90 mol % of thetotal lipid present in the lipid nanoparticle.

In some aspects, the lipid nanoparticle can further comprise anon-cationic lipid. Non-ionic lipids include amphipathic lipids, neutrallipids and anionic lipids. Accordingly, the non-cationic lipid can be aneutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipidsare typically employed to enhance fusogenicity.

Exemplary non-cationic lipids envisioned for use in the methods andcompositions as disclosed herein are described in InternationalApplication PCT/US2018/050042, filed on Sep. 7, 2018, andPCT/US2018/064242, filed on Dec. 6, 2018 which is incorporated herein inits entirety. Exemplary non-cationic lipids are described inInternational Application Publication WO2017/099823 and US patentpublication US2018/0028664, the contents of both of which areincorporated herein by reference in their entirety.

The non-cationic lipid can comprise 0-30% (mol) of the total lipidpresent in the lipid nanoparticle. For example, the non-cationic lipidcontent is 5-20% (mol) or 10-15% (mol) of the total lipid present in thelipid nanoparticle. In various embodiments, the molar ratio of ionizablelipid to the neutral lipid ranges from about 2:1 to about 8:1.

In some embodiments, the lipid nanoparticles do not comprise anyphospholipids. In some aspects, the lipid nanoparticle can furthercomprise a component, such as a sterol, to provide membrane integrity.

One exemplary sterol that can be used in the lipid nanoparticle ischolesterol and derivatives thereof. Exemplary cholesterol derivativesare described in International application WO2009/127060 and US patentpublication US2010/0130588, contents of both of which are incorporatedherein by reference in their entirety.

The component providing membrane integrity, such as a sterol, cancomprise 0-50% (mol) of the total lipid present in the lipidnanoparticle. In some embodiments, such a component is 20-50% (mol)30-40% (mol) of the total lipid content of the lipid nanoparticle.

In some aspects, the lipid nanoparticle can further comprise apolyethylene glycol (PEG) or a conjugated lipid molecule. Generally,these are used to inhibit aggregation of lipid nanoparticles and/orprovide steric stabilization. Exemplary conjugated lipids include, butare not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipidconjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates),cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In someembodiments, the conjugated lipid molecule is a PEG-lipid conjugate, forexample, a (methoxy polyethylene glycol)-conjugated lipid. ExemplaryPEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol(DAG) (such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)),PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), apegylated phosphatidylethanoloamine (PEG-PE), PEG succinatediacylglycerol (PEGS-DAG) (such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam,N-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or amixture thereof. Additional exemplary PEG-lipid conjugates aredescribed, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591,US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058,US2011/0117125, US2010/0130588, US2016/0376224, and US2017/0119904, thecontents of all of which are incorporated herein by reference in theirentirety.

In some embodiments, a PEG-lipid is a compound as defined inUS2018/0028664, the content of which is incorporated herein by referencein its entirety. In some embodiments, a PEG-lipid is disclosed inUS20150376115 or in US2016/0376224, the content of both of which isincorporated herein by reference in its entirety.

The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl,PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, orPEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG,PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol,PEG-dilaurylglycamide, PEG-dimyristylglycamide,PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol(1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB(3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether),and1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]. In some examples, the PEG-lipid can be selected from thegroup consisting of PEG-DMG,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000],

Lipids conjugated with a molecule other than a PEG can also be used inplace of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates,polyamide-lipid conjugates (such as ATTA-lipid conjugates), andcationic-polymer lipid (CPL) conjugates can be used in place of or inaddition to the PEG-lipid. Exemplary conjugated lipids, i.e.,PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationicpolymer-lipids are described in the International patent applicationpublications WO1996/010392, WO1998/051278, WO2002/087541, WO2005/026372,WO2008/147438, WO2009/086558, WO2012/000104, WO2017/117528,WO2017/099823, WO2015/199952, WO2017/004143, WO2015/095346,WO2012/000104, WO2012/000104, and WO2010/006282, US patent applicationpublications US2003/0077829, US2005/0175682, US2008/0020058,US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115,US2016/0376224, US2016/0317458, US2013/0303587, US2013/0303587, andUS20110123453, and US patents U.S. Pat. Nos. 5,885,613, 6,287,591,6,320,017, and 6,586,559, the contents of all of which are incorporatedherein by reference in their entirety.

In some embodiments, the one or more additional compound can be atherapeutic agent. The therapeutic agent can be selected from any classsuitable for the therapeutic objective. In other words, the therapeuticagent can be selected from any class suitable for the therapeuticobjective. In other words, the therapeutic agent can be selectedaccording to the treatment objective and biological action desired. Forexample, if the ceDNA within the LNP is useful for treating hemophiliaA, the additional compound can be an anti-hemophilia A agent (e.g., achemotherapeutic agent, or other hemophilia A therapy (including, butnot limited to, a small molecule or an antibody). In another example, ifthe LNP containing the ceDNA is useful for treating an infection, theadditional compound can be an antimicrobial agent (e.g., an antibioticor antiviral compound). In yet another example, if the LNP containingthe ceDNA is useful for treating an immune disease or disorder, theadditional compound can be a compound that modulates an immune response(e.g., an immunosuppressant, immunostimulatory compound, or compoundmodulating one or more specific immune pathways). In some embodiments,different cocktails of different lipid nanoparticles containingdifferent compounds, such as a ceDNA encoding a different protein or adifferent compound, such as a therapeutic may be used in thecompositions and methods of the invention.

In some embodiments, the additional compound is an immune modulatingagent. For example, the additional compound is an immunosuppressant. Insome embodiments, the additional compound is immune stimulatory agent.Also provided herein is a pharmaceutical composition comprising thelipid nanoparticle-encapsulated insect-cell produced, or a syntheticallyproduced ceDNA vector for expression of FVIII protein as describedherein and a pharmaceutically acceptable carrier or excipient.

In some aspects, the disclosure provides for a lipid nanoparticleformulation further comprising one or more pharmaceutical excipients. Insome embodiments, the lipid nanoparticle formulation further comprisessucrose, tris, trehalose and/or glycine.

The ceDNA vector can be complexed with the lipid portion of the particleor encapsulated in the lipid position of the lipid nanoparticle. In someembodiments, the ceDNA can be fully encapsulated in the lipid positionof the lipid nanoparticle, thereby protecting it from degradation by anuclease, e.g., in an aqueous solution. In some embodiments, the ceDNAin the lipid nanoparticle is not substantially degraded after exposureof the lipid nanoparticle to a nuclease at 37° C. for at least about 20,30, 45, or 60 minutes. In some embodiments, the ceDNA in the lipidnanoparticle is not substantially degraded after incubation of theparticle in serum at 37° C. for at least about 30, 45, or 60 minutes orat least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, or 36 hours.

In certain embodiments, the lipid nanoparticles are substantiallynon-toxic to a subject, e.g., to a mammal such as a human In someaspects, the lipid nanoparticle formulation is a lyophilized powder.

In some embodiments, lipid nanoparticles are solid core particles thatpossess at least one lipid bilayer. In other embodiments, the lipidnanoparticles have a non-bilayer structure, i.e., a non-lamellar (i.e.,non-bilayer) morphology. Without limitations, the non-bilayer morphologycan include, for example, three dimensional tubes, rods, cubicsymmetries, etc. For example, the morphology of the lipid nanoparticles(lamellar vs. non-lamellar) can readily be assessed and characterizedusing, e.g., Cryo-TEM analysis as described in US2010/0130588, thecontent of which is incorporated herein by reference in its entirety.

In some further embodiments, the lipid nanoparticles having anon-lamellar morphology are electron dense. In some aspects, thedisclosure provides for a lipid nanoparticle that is either unilamellaror multilamellar in structure. In some aspects, the disclosure providesfor a lipid nanoparticle formulation that comprises multi-vesicularparticles and/or foam-based particles.

By controlling the composition and concentration of the lipidcomponents, one can control the rate at which the lipid conjugateexchanges out of the lipid particle and, in turn, the rate at which thelipid nanoparticle becomes fusogenic. In addition, other variablesincluding, e.g., pH, temperature, or ionic strength, can be used to varyand/or control the rate at which the lipid nanoparticle becomesfusogenic. Other methods which can be used to control the rate at whichthe lipid nanoparticle becomes fusogenic will be apparent to those ofordinary skill in the art based on this disclosure. It will also beapparent that by controlling the composition and concentration of thelipid conjugate, one can control the lipid particle size.

The pKa of formulated cationic lipids can be correlated with theeffectiveness of the LNPs for delivery of nucleic acids (see Jayaramanet al., Angewandte Chemie, International Edition (2012), 51(34),8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), bothof which are incorporated by reference in their entirety). The preferredrange of pKa is ˜5 to ˜7. The pKa of the cationic lipid can bedetermined in lipid nanoparticles using an assay based on fluorescenceof 2-(p-toluidino)-6-napthalene sulfonic acid (TNS).

VIII. Methods of Use

A ceDNA vector for expression of FVIII protein as disclosed herein canalso be used in a method for the delivery of a nucleotide sequence ofinterest (e.g., encoding FVIII protein) to a target cell (e.g., a hostcell). The method may in particular be a method for delivering FVIIIprotein to a cell of a subject in need thereof and treating hemophiliaA. The invention allows for the in vivo expression of FVIII proteinencoded in the ceDNA vector in a cell in a subject such that therapeuticeffect of the expression of FVIII protein occurs. These results are seenwith both in vivo and in vitro modes of ceDNA vector delivery.

In addition, the invention provides a method for the delivery of FVIIIprotein in a cell of a subject in need thereof, comprising multipleadministrations of the ceDNA vector of the invention encoding said FVIIIprotein. Since the ceDNA vector of the invention does not induce animmune response like that typically observed against encapsidated viralvectors, such a multiple administration strategy will likely havegreater success in a ceDNA-based system. The ceDNA vector areadministered in sufficient amounts to transfect the cells of a desiredtissue and to provide sufficient levels of gene transfer and expressionof the FVIII protein without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, retinal administration (e.g., subretinal injection,suprachoroidal injection or intravitreal injection), intravenous (e.g.,in a liposome formulation), direct delivery to the selected organ (e.g.,any one or more tissues selected from: liver, kidneys, gallbladder,prostate, adrenal gland, heart, intestine, lung, and stomach),intramuscular, and other parental routes of administration. Routes ofadministration may be combined, if desired.

Delivery of a ceDNA vector for expression of FVIII protein as describedherein is not limited to delivery of the expressed FVIII protein. Forexample, conventionally produced (e.g., using a cell-based productionmethod (e.g., insect-cell production methods) or synthetically producedceDNA vectors as described herein may be used with other deliverysystems provided to provide a portion of the gene therapy. Onenon-limiting example of a system that may be combined with the ceDNAvectors in accordance with the present disclosure includes systems whichseparately deliver one or more co-factors or immune suppressors foreffective gene expression of the ceDNA vector expressing the FVIIIprotein.

The invention also provides for a method of treating hemophilia A in asubject comprising introducing into a target cell in need thereof (inparticular a muscle cell or tissue) of the subject a therapeuticallyeffective amount of a ceDNA vector, optionally with a pharmaceuticallyacceptable carrier. While the ceDNA vector can be introduced in thepresence of a carrier, such a carrier is not required. The ceDNA vectorselected comprises a nucleotide sequence encoding an FVIII proteinuseful for treating hemophilia A. In particular, the ceDNA vector maycomprise a desired FVIII protein sequence operably linked to controlelements capable of directing transcription of the desired FVIII proteinencoded by the exogenous DNA sequence when introduced into the subject.The ceDNA vector can be administered via any suitable route as providedabove, and elsewhere herein.

The compositions and vectors provided herein can be used to deliver anFVIII protein for various purposes. In some embodiments, the transgeneencodes an FVIII protein that is intended to be used for researchpurposes, e.g., to create a somatic transgenic animal model harboringthe transgene, e.g., to study the function of the FVIII protein product.In another example, the transgene encodes an FVIII protein that isintended to be used to create an animal model of hemophilia A. In someembodiments, the encoded FVIII protein is useful for the treatment orprevention of hemophilia A states in a mammalian subject. The FVIIIprotein can be transferred (e.g., expressed in) to a patient in asufficient amount to treat hemophilia A associated with reducedexpression, lack of expression or dysfunction of the gene.

In principle, the expression cassette can include a nucleic acid or anytransgene that encodes an FVIII protein that is either reduced or absentdue to a mutation or which conveys a therapeutic benefit whenoverexpressed is considered to be within the scope of the invention.Preferably, noninserted bacterial DNA is not present and preferably nobacterial DNA is present in the ceDNA compositions provided herein.

A ceDNA vector is not limited to one species of ceDNA vector. As such,in another aspect, multiple ceDNA vectors expressing different proteinsor the same FVIII protein but operatively linked to different promotersor cis-regulatory elements can be delivered simultaneously orsequentially to the target cell, tissue, organ, or subject. Therefore,this strategy can allow for the gene therapy or gene delivery ofmultiple proteins simultaneously. It is also possible to separatedifferent portions of a FVIII protein into separate ceDNA vectors (e.g.,different domains and/or co-factors required for functionality of aFVIII protein) which can be administered simultaneously or at differenttimes, and can be separately regulatable, thereby adding an additionallevel of control of expression of a FVIII protein. Delivery can also beperformed multiple times and, importantly for gene therapy in theclinical setting, in subsequent increasing or decreasing doses, giventhe lack of an anti-capsid host immune response due to the absence of aviral capsid. It is anticipated that no anti-capsid response will occuras there is no capsid.

The invention also provides for a method of treating hemophilia A in asubject comprising introducing into a target cell in need thereof (inparticular a muscle cell or tissue) of the subject a therapeuticallyeffective amount of a ceDNA vector as disclosed herein, optionally witha pharmaceutically acceptable carrier. While the ceDNA vector can beintroduced in the presence of a carrier, such a carrier is not required.The ceDNA vector implemented comprises a nucleotide sequence of interestuseful for treating the hemophilia A. In particular, the ceDNA vectormay comprise a desired exogenous DNA sequence operably linked to controlelements capable of directing transcription of the desired polypeptide,protein, or oligonucleotide encoded by the exogenous DNA sequence whenintroduced into the subject. The ceDNA vector can be administered viaany suitable route as provided above, and elsewhere herein.

IX. Methods of Delivering ceDNA Vectors for FVIII Protein Production

In some embodiments, a ceDNA vector for expression of FVIII protein canbe delivered to a target cell in vitro or in vivo by various suitablemethods. ceDNA vectors alone can be applied or injected. CeDNA vectorscan be delivered to a cell without the help of a transfection reagent orother physical means. Alternatively, ceDNA vectors for expression ofFVIII protein can be delivered using any art-known transfection reagentor other art-known physical means that facilitates entry of DNA into acell, e.g., liposomes, alcohols, polylysine-rich compounds,arginine-rich compounds, calcium phosphate, microvesicles,microinjection, electroporation and the like.

The ceDNA vectors for expression of FVIII protein as disclosed hereincan efficiently target cell and tissue-types that are normally difficultto transduce with conventional AAV virions using various deliveryreagent.

One aspect of the technology described herein relates to a method ofdelivering an FVIII protein to a cell. Typically, for in vivo and invitro methods, a ceDNA vector for expression of FVIII protein asdisclosed herein may be introduced into the cell using the methods asdisclosed herein, as well as other methods known in the art. A ceDNAvector for expression of FVIII protein as disclosed herein arepreferably administered to the cell in a biologically-effective amount.If the ceDNA vector is administered to a cell in vivo (e.g., to asubject), a biologically-effective amount of the ceDNA vector is anamount that is sufficient to result in transduction and expression ofthe FVIII protein in a target cell.

Exemplary modes of administration of a ceDNA vector for expression ofFVIII protein as disclosed herein includes oral, rectal, transmucosal,intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal,intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous,subcutaneous, intradermal, intracranial, intramuscular [includingadministration to skeletal, diaphragm and/or cardiac muscle],intrapleural, intracerebral, and intraarticular). Administration can besystemically or direct delivery to the liver or elsewhere (e.g., anykidneys, gallbladder, prostate, adrenal gland, heart, intestine, lung,and stomach).

Administration can be topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intralymphatic, and the like, as well as direct tissue or organinjection (e.g., but not limited to, liver, but also to eye, muscles,including skeletal muscle, cardiac muscle, diaphragm muscle, or brain).

Administration of the ceDNA vector can be to any site in a subject,including, without limitation, a site selected from the group consistingof the liver and/or also eyes, brain, a skeletal muscle, a smoothmuscle, the heart, the diaphragm, the airway epithelium, the kidney, thespleen, the pancreas, the skin.

The most suitable route in any given case will depend on the nature andseverity of the condition being treated, ameliorated, and/or preventedand on the nature of the particular ceDNA vector that is being used.Additionally, ceDNA permits one to administer more than one FVIIIprotein in a single vector, or multiple ceDNA vectors (e.g. a ceDNAcocktail).

A. Intramuscular Administration of a ceDNA Vector

In some embodiments, a method of treating a disease in a subjectcomprises introducing into a target cell in need thereof (in particulara muscle cell or tissue) of the subject a therapeutically effectiveamount of a ceDNA vector encoding an FVIII protein, optionally with apharmaceutically acceptable carrier. In some embodiments, the ceDNAvector for expression of FVIII protein is administered to a muscletissue of a subject.

In some embodiments, administration of the ceDNA vector can be to anysite in a subject, including, without limitation, a site selected fromthe group consisting of a skeletal muscle, a smooth muscle, the heart,the diaphragm, or muscles of the eye.

Administration of a ceDNA vector for expression of FVIII protein asdisclosed herein to a skeletal muscle according to the present inventionincludes but is not limited to administration to the skeletal muscle inthe limbs (e.g., upper arm, lower arm, upper leg, and/or lower leg),back, neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum,and/or digits. The ceDNA as disclosed herein vector can be delivered toskeletal muscle by intravenous administration, intra-arterialadministration, intraperitoneal administration, limb perfusion,(optionally, isolated limb perfusion of a leg and/or arm; see, e.g.Arruda et al., (2005) Blood 105: 3458-3464), and/or direct intramuscularinjection. In particular embodiments, the ceDNA vector as disclosedherein is administered to the liver, eye, a limb (arm and/or leg) of asubject (e.g., a subject with muscular dystrophy such as DMD) by limbperfusion, optionally isolated limb perfusion (e.g., by intravenous orintra-articular administration. In embodiments, the ceDNA vector asdisclosed herein can be administered without employing “hydrodynamic”techniques.

For instance, tissue delivery (e.g., to retina) of conventional viralvectors is often enhanced by hydrodynamic techniques (e.g.,intravenous/intravenous administration in a large volume), whichincrease pressure in the vasculature and facilitate the ability of theviral vector to cross the endothelial cell barrier. In particularembodiments, the ceDNA vectors described herein can be administered inthe absence of hydrodynamic techniques such as high volume infusionsand/or elevated intravascular pressure (e.g., greater than normalsystolic pressure, for example, less than or equal to a 5%, 10%, 15%,20%, 25% increase in intravascular pressure over normal systolicpressure). Such methods may reduce or avoid the side effects associatedwith hydrodynamic techniques such as edema, nerve damage and/orcompartment syndrome.

Furthermore, a composition comprising a ceDNA vector for expression ofFVIII protein as disclosed herein that is administered to a skeletalmuscle can be administered to a skeletal muscle in the limbs (e.g.,upper arm, lower arm, upper leg, and/or lower leg), back, neck, head(e.g., tongue), thorax, abdomen, pelvis/perineum, and/or digits.Suitable skeletal muscles include but are not limited to abductor digitiminimi (in the hand), abductor digiti minimi (in the foot), abductorhallucis, abductor ossis metatarsi quinti, abductor pollicis brevis,abductor pollicis longus, adductor brevis, adductor hallucis, adductorlongus, adductor magnus, adductor pollicis, anconeus, anterior scalene,articularis genus, biceps brachii, biceps femoris, brachialis,brachioradialis, buccinator, coracobrachialis, corrugator supercilii,deltoid, depressor anguli oris, depressor labii inferioris, digastric,dorsal interossei (in the hand), dorsal interossei (in the foot),extensor carpi radialis brevis, extensor carpi radialis longus, extensorcarpi ulnaris, extensor digiti minimi, extensor digitorum, extensordigitorum brevis, extensor digitorum longus, extensor hallucis brevis,extensor hallucis longus, extensor indicis, extensor pollicis brevis,extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris,flexor digiti minimi brevis (in the hand), flexor digiti minimi brevis(in the foot), flexor digitorum brevis, flexor digitorum longus, flexordigitorum profundus, flexor digitorum superficialis, flexor hallucisbrevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicislongus, frontalis, gastrocnemius, geniohyoid, gluteus maximus, gluteusmedius, gluteus minimus, gracilis, iliocostalis cervicis, iliocostalislumborum, iliocostalis thoracis, illiacus, inferior gemellus, inferioroblique, inferior rectus, infraspinatus, interspinalis, intertransversi,lateral pterygoid, lateral rectus, latissimus dorsi, levator angulioris, levator labii superioris, levator labii superioris alaeque nasi,levator palpebrae superioris, levator scapulae, long rotators,longissimus capitis, longissimus cervicis, longissimus thoracis, longuscapitis, longus colli, lumbricals (in the hand), lumbricals (in thefoot), masseter, medial pterygoid, medial rectus, middle scalene,multifidus, mylohyoid, obliquus capitis inferior, obliquus capitissuperior, obturator externus, obturator internus, occipitalis, omohyoid,opponens digiti minimi, opponens pollicis, orbicularis oculi,orbicularis oris, palmar interossei, palmaris brevis, palmaris longus,pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneuslongus, peroneus tertius, piriformis, plantar interossei, plantaris,platysma, popliteus, posterior scalene, pronator quadratus, pronatorteres, psoas major, quadratus femoris, quadratus plantae, rectus capitisanterior, rectus capitis lateralis, rectus capitis posterior major,rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboidminor, risorius, sartorius, scalenus minimus, semimembranosus,semispinalis capitis, semispinalis cervicis, semispinalis thoracis,semitendinosus, serratus anterior, short rotators, soleus, spinaliscapitis, spinalis cervicis, spinalis thoracis, splenius capitis,splenius cervicis, sternocleidomastoid, sternohyoid, sternothyroid,stylohyoid, subclavius, subscapularis, superior gemellus, superioroblique, superior rectus, supinator, supraspinatus, temporalis, tensorfascia lata, teres major, teres minor, thoracis, thyrohyoid, tibialisanterior, tibialis posterior, trapezius, triceps brachii, vastusintermedius, vastus lateralis, vastus medialis, zygomaticus major, andzygomaticus minor, and any other suitable skeletal muscle as known inthe art.

Administration of a ceDNA vector for expression of FVIII protein asdisclosed herein to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration. In some embodiments, delivery ofan expressed transgene from the ceDNA vector to a target tissue can alsobe achieved by delivering a synthetic depot comprising the ceDNA vector,where a depot comprising the ceDNA vector is implanted into skeletal,smooth, cardiac and/or diaphragm muscle tissue or the muscle tissue canbe contacted with a film or other matrix comprising the ceDNA vector asdescribed herein. Such implantable matrices or substrates are describedin U.S. Pat. No. 7,201,898.

Administration of a ceDNA vector for expression of FVIII protein asdisclosed herein to cardiac muscle includes administration to the leftatrium, right atrium, left ventricle, right ventricle and/or septum. TheceDNA vector as described herein can be delivered to cardiac muscle byintravenous administration, intra-arterial administration such asintra-aortic administration, direct cardiac injection (e.g., into leftatrium, right atrium, left ventricle, right ventricle), and/or coronaryartery perfusion.

Administration of a ceDNA vector for expression of FVIII protein asdisclosed herein to smooth muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration. In one embodiment,administration can be to endothelial cells present in, near, and/or onsmooth muscle. Non-limiting examples of smooth muscles include the irisof the eye, bronchioles of the lung, laryngeal muscles (vocal cords),muscular layers of the stomach, esophagus, small and large intestine ofthe gastrointestinal tract, ureter, detrusor muscle of the urinarybladder, uterine myometrium, penis, or prostate gland.

In some embodiments, of a ceDNA vector for expression of FVIII proteinas disclosed herein is administered to skeletal muscle, diaphragm muscleand/or cardiac muscle. In representative embodiments, a ceDNA vectoraccording to the present invention is used to treat and/or preventdisorders of skeletal, cardiac and/or diaphragm muscle.

Specifically, it is contemplated that a composition comprising a ceDNAvector for expression of FVIII protein as disclosed herein can bedelivered to one or more muscles of the eye (e.g., Lateral rectus,Medial rectus, Superior rectus, Inferior rectus, Superior oblique,Inferior oblique), facial muscles (e.g., Occipitofrontalis muscle,Temporoparietalis muscle, Procerus muscle, Nasalis muscle, Depressorsepti nasi muscle, Orbicularis oculi muscle, Corrugator superciliimuscle, Depressor supercilii muscle, Auricular muscles, Orbicularis orismuscle, Depressor anguli oris muscle, Risorius, Zygomaticus majormuscle, Zygomaticus minor muscle, Levator labii superioris, Levatorlabii superioris alaeque nasi muscle, Depressor labii inferioris muscle,Levator anguli oris, Buccinator muscle, Mentalis) or tongue muscles(e.g., genioglossus, hyoglossus, chondroglossus, styloglossus,palatoglossus, superior longitudinal muscle, inferior longitudinalmuscle, the vertical muscle, and the transverse muscle).

(i) Intramuscular Injection:

In some embodiments, a composition comprising a ceDNA vector forexpression of FVIII protein as disclosed herein can be injected into oneor more sites of a given muscle, for example, skeletal muscle (e.g.,deltoid, vastus lateralis, ventrogluteal muscle of dorsogluteal muscle,or anterolateral thigh for infants) in a subject using a needle. Thecomposition comprising ceDNA can be introduced to other subtypes ofmuscle cells. Non-limiting examples of muscle cell subtypes includeskeletal muscle cells, cardiac muscle cells, smooth muscle cells and/ordiaphragm muscle cells.

Methods for intramuscular injection are known to those of skill in theart and as such are not described in detail herein. However, whenperforming an intramuscular injection, an appropriate needle size shouldbe determined based on the age and size of the patient, the viscosity ofthe composition, as well as the site of injection. Table 8 providesguidelines for exemplary sites of injection and corresponding needlesize:

TABLE 8 Guidelines for intramuscular injection in human patients MaximumInjection Needle Needle volume of Site Gauge Size compositionVentrogluteal Aqueous Thin adult: 15 to 25 mm 3 mL site solutions:Average adult: 25 mm (gluteus 20-25 gauge Larger adult medius Viscous oroil- (over 150 lbs): and gluteus based solution: 25 to 38 mm. minimus)18-21 gauge Children and infants: will require a smaller needle VastusAqueous Adult: 25 mm to 38 mm 3 mL lateralis solutions: 20-25 gaugeViscous or oil- based solution: 18-21 gauge Children/infants: 22 to 25gauge Deltoid 22 to 25 gauge Males: 1 mL 130-2601bs: 25 mm Females: <130lbs: 16 mm 130-200 lbs: 25 mm >2001bs: 38 mm

In certain embodiments, a ceDNA vector for expression of FVIII proteinas disclosed herein is formulated in a small volume, for example, anexemplary volume as outlined in Table 8 for a given subject. In someembodiments, the subject can be administered a general or localanesthetic prior to the injection, if desired. This is particularlydesirable if multiple injections are required or if a deeper muscle isinjected, rather than the common injection sites noted above.

In some embodiments, intramuscular injection can be combined withelectroporation, delivery pressure or the use of transfection reagentsto enhance cellular uptake of the ceDNA vector.

(ii) Transfection Reagents:

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is formulated in compositions comprising one or moretransfection reagents to facilitate uptake of the vectors into myotubesor muscle tissue. Thus, in one embodiment, the nucleic acids describedherein are administered to a muscle cell, myotube or muscle tissue bytransfection using methods described elsewhere herein.

(iii) Electroporation:

In certain embodiments, a ceDNA vector for expression of FVIII proteinas disclosed herein is administered in the absence of a carrier tofacilitate entry of ceDNA into the cells, or in a physiologically inertpharmaceutically acceptable carrier (i.e., any carrier that does notimprove or enhance uptake of the capsid free, non-viral vectors into themyotubes). In such embodiments, the uptake of the capsid free, non-viralvector can be facilitated by electroporation of the cell or tissue.

Cell membranes naturally resist the passage of extracellular into thecell cytoplasm. One method for temporarily reducing this resistance is“electroporation”, where electrical fields are used to create pores incells without causing permanent damage to the cells. These pores arelarge enough to allow DNA vectors, pharmaceutical drugs, DNA, and otherpolar compounds to gain access to the interior of the cell. With time,the pores in the cell membrane close and the cell once again becomesimpermeable.

Electroporation can be used in both in vitro and in vivo applications tointroduce e.g., exogenous DNA into living cells. In vitro applicationstypically mix a sample of live cells with the composition comprisinge.g., DNA. The cells are then placed between electrodes such as parallelplates and an electrical field is applied to the cell/compositionmixture.

There are a number of methods for in vivo electroporation; electrodescan be provided in various configurations such as, for example, acaliper that grips the epidermis overlying a region of cells to betreated. Alternatively, needle-shaped electrodes may be inserted intothe tissue, to access more deeply located cells. In either case, afterthe composition comprising e.g., nucleic acids are injected into thetreatment region, the electrodes apply an electrical field to theregion. In some electroporation applications, this electric fieldcomprises a single square wave pulse on the order of 100 to 500 V/cm. ofabout 10 to 60 ms duration. Such a pulse may be generated, for example,in known applications of the Electro Square Porator T820, made by theBTX Division of Genetronics, Inc.

Typically, successful uptake of e.g., nucleic acids occurs only if themuscle is electrically stimulated immediately, or shortly afteradministration of the composition, for example, by injection into themuscle.

In certain embodiments, electroporation is achieved using pulses ofelectric fields or using low voltage/long pulse treatment regimens(e.g., using a square wave pulse electroporation system). Exemplarypulse generators capable of generating a pulsed electric field include,for example, the ECM600, which can generate an exponential wave form,and the ElectroSquarePorator (T820), which can generate a square waveform, both of which are available from BTX, a division of Genetronics,Inc. (San Diego, Calif.). Square wave electroporation systems delivercontrolled electric pulses that rise quickly to a set voltage, stay atthat level for a set length of time (pulse length), and then quicklydrop to zero.

In some embodiments, a local anesthetic is administered, for example, byinjection at the site of treatment to reduce pain that may be associatedwith electroporation of the tissue in the presence of a compositioncomprising a capsid free, non-viral vector as described herein. Inaddition, one of skill in the art will appreciate that a dose of thecomposition should be chosen that minimizes and/or prevents excessivetissue damage resulting in fibrosis, necrosis or inflammation of themuscle.

(iv) Delivery Pressure:

In some embodiments, delivery of a ceDNA vector for expression of FVIIIprotein as disclosed herein to muscle tissue is facilitated by deliverypressure, which uses a combination of large volumes and rapid injectioninto an artery supplying a limb (e.g., iliac artery). This mode ofadministration can be achieved through a variety of methods that involveinfusing limb vasculature with a composition comprising a ceDNA vector,typically while the muscle is isolated from the systemic circulationusing a tourniquet of vessel clamps. In one method, the composition iscirculated through the limb vasculature to permit extravasation into thecells. In another method, the intravascular hydrodynamic pressure isincreased to expand vascular beds and increase uptake of the ceDNAvector into the muscle cells or tissue. In one embodiment, the ceDNAcomposition is administered into an artery.

(v) Lipid Nanoparticle Compositions:

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein for intramuscular delivery are formulated in acomposition comprising a liposome as described elsewhere herein.

(vi) Systemic Administration of a ceDNA Vector Targeted to MuscleTissue:

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein is formulated to be targeted to the muscle via indirectdelivery administration, where the ceDNA is transported to the muscle asopposed to the liver. Accordingly, the technology described hereinencompasses indirect administration of compositions comprising a ceDNAvector for expression of FVIII protein as disclosed herein to muscletissue, for example, by systemic administration. Such compositions canbe administered topically, intravenously (by bolus or continuousinfusion), intracellular injection, intratissue injection, orally, byinhalation, intraperitoneally, subcutaneously, intracavity, and can bedelivered by peristaltic means, if desired, or by other means known bythose skilled in the art. The agent can be administered systemically,for example, by intravenous infusion, if so desired.

In some embodiments, uptake of a ceDNA vector for expression of FVIIIprotein as disclosed herein into muscle cells/tissue is increased byusing a targeting agent or moiety that preferentially directs the vectorto muscle tissue. Thus, in some embodiments, a capsid free, ceDNA vectorcan be concentrated in muscle tissue as compared to the amount of capsidfree ceDNA vectors present in other cells or tissues of the body.

In some embodiments, the composition comprising a ceDNA vector forexpression of FVIII protein as disclosed herein further comprises atargeting moiety to muscle cells. In other embodiments, the expressedgene product comprises a targeting moiety specific to the tissue inwhich it is desired to act. The targeting moiety can include anymolecule, or complex of molecules, which is/are capable of targeting,interacting with, coupling with, and/or binding to an intracellular,cell surface, or extracellular biomarker of a cell or tissue. Thebiomarker can include, for example, a cellular protease, a kinase, aprotein, a cell surface receptor, a lipid, and/or fatty acid. Otherexamples of biomarkers that the targeting moieties can target, interactwith, couple with, and/or bind to include molecules associated with aparticular disease. For example, the biomarkers can include cell surfacereceptors implicated in cancer development, such as epidermal growthfactor receptor and transferrin receptor. The targeting moieties caninclude, but are not limited to, synthetic compounds, natural compoundsor products, macromolecular entities, bioengineered molecules (e.g.,polypeptides, lipids, polynucleotides, antibodies, antibody fragments),and small entities (e.g., small molecules, neurotransmitters,substrates, ligands, hormones and elemental compounds) that bind tomolecules expressed in the target muscle tissue.

In certain embodiments, the targeting moiety may further comprise areceptor molecule, including, for example, receptors, which naturallyrecognize a specific desired molecule of a target cell. Such receptormolecules include receptors that have been modified to increase theirspecificity of interaction with a target molecule, receptors that havebeen modified to interact with a desired target molecule not naturallyrecognized by the receptor, and fragments of such receptors (see, e.g.,Skerra, 2000, J. Molecular Recognition, 13:167-187). A preferredreceptor is a chemokine receptor. Exemplary chemokine receptors havebeen described in, for example, Lapidot et al, 2002, Exp Hematol,30:973-81 and Onuffer et al., 2002, Trends Pharmacol Sci, 23:459-67.

In other embodiments, the additional targeting moiety may comprise aligand molecule, including, for example, ligands which naturallyrecognize a specific desired receptor of a target cell, such as aTransferrin (Tf) ligand. Such ligand molecules include ligands that havebeen modified to increase their specificity of interaction with a targetreceptor, ligands that have been modified to interact with a desiredreceptor not naturally recognized by the ligand, and fragments of suchligands.

In still other embodiments, the targeting moiety may comprise anaptamer. Aptamers are oligonucleotides that are selected to bindspecifically to a desired molecular structure of the target cell.Aptamers typically are the products of an affinity selection processsimilar to the affinity selection of phage display (also known as invitro molecular evolution). The process involves performing severaltandem iterations of affinity separation, e.g., using a solid support towhich the diseased immunogen is bound, followed by polymerase chainreaction (PCR) to amplify nucleic acids that bound to the immunogens.Each round of affinity separation thus enriches the nucleic acidpopulation for molecules that successfully bind the desired immunogen.In this manner, a random pool of nucleic acids may be “educated” toyield aptamers that specifically bind target molecules. Aptamerstypically are RNA, but may be DNA or analogs or derivatives thereof,such as, without limitation, peptide nucleic acids (PNAs) andphosphorothioate nucleic acids.

In some embodiments, the targeting moiety can comprise aphoto-degradable ligand (i.e., a ‘caged’ ligand) that is released, forexample, from a focused beam of light such that the capsid free,non-viral vectors or the gene product are targeted to a specific tissue.

It is also contemplated herein that the compositions be delivered tomultiple sites in one or more muscles of the subject. That is,injections can be in at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55, at least 60, at least 65, at least70, at least 75, at least 80, at least 85, at least 90, at least 95, atleast 100 injections sites. Such sites can be spread over the area of asingle muscle or can be distributed among multiple muscles.

B. Administration of the ceDNA Vector for Expression of FVIII Protein toNon-Muscle Locations

In another embodiment, a ceDNA vector for expression of FVIII protein isadministered to the liver. The ceDNA vector may also be administered todifferent regions of the eye such as the cornea and/or optic nerve TheceDNA vector may also be introduced into the spinal cord, brainstem(medulla oblongata, pons), midbrain (hypothalamus, thalamus,epithalamus, pituitary gland, substantia nigra, pineal gland),cerebellum, telencephalon (corpus striatum, cerebrum including theoccipital, temporal, parietal and frontal lobes, cortex, basal ganglia,hippocampus and portaamygdala), limbic system, neocortex, corpusstriatum, cerebrum, and inferior colliculus. The ceDNA vector may bedelivered into the cerebrospinal fluid (e.g., by lumbar puncture). TheceDNA vector for expression of FVIII protein may further be administeredintravascularly to the CNS in situations in which the blood-brainbarrier has been perturbed (e.g., brain tumor or cerebral infarct).

In some embodiments, the ceDNA vector for expression of FVIII proteincan be administered to the desired region(s) of the eye by any routeknown in the art, including but not limited to, intrathecal,intra-ocular, intracerebral, intraventricular, intravenous (e.g., in thepresence of a sugar such as mannitol), intranasal, intra-aural,intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) andperi-ocular (e.g., sub-Tenon's region) delivery as well as intramusculardelivery with retrograde delivery to motor neurons.

In some embodiments, the ceDNA vector for expression of FVIII protein isadministered in a liquid formulation by direct injection (e.g.,stereotactic injection) to the desired region or compartment in the CNS.In other embodiments, the ceDNA vector can be provided by topicalapplication to the desired region or by intra-nasal administration of anaerosol formulation. Administration to the eye may be by topicalapplication of liquid droplets. As a further alternative, the ceDNAvector can be administered as a solid, slow-release formulation (see,e.g., U.S. Pat. No. 7,201,898). In yet additional embodiments, the ceDNAvector can used for retrograde transport to treat, ameliorate, and/orprevent diseases and disorders involving motor neurons (e.g.,amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA),etc.). For example, the ceDNA vector can be delivered to muscle tissuefrom which it can migrate into neurons.

C. Ex Vivo Treatment

In some embodiments, cells are removed from a subject, a ceDNA vectorfor expression of FVIII protein as disclosed herein is introducedtherein, and the cells are then replaced back into the subject. Methodsof removing cells from subject for treatment ex vivo, followed byintroduction back into the subject are known in the art (see, e.g., U.S.Pat. No. 5,399,346; the disclosure of which is incorporated herein inits entirety). Alternatively, a ceDNA vector is introduced into cellsfrom another subject, into cultured cells, or into cells from any othersuitable source, and the cells are administered to a subject in needthereof.

Cells transduced with a ceDNA vector for expression of FVIII protein asdisclosed herein are preferably administered to the subject in a“therapeutically-effective amount” in combination with a pharmaceuticalcarrier. Those skilled in the art will appreciate that the therapeuticeffects need not be complete or curative, as long as some benefit isprovided to the subject.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein can encode an FVIII protein as described herein(sometimes called a transgene or heterologous nucleotide sequence) thatis to be produced in a cell in vitro, ex vivo, or in vivo. For example,in contrast to the use of the ceDNA vectors described herein in a methodof treatment as discussed herein, in some embodiments a ceDNA vector forexpression of FVIII protein may be introduced into cultured cells andthe expressed FVIII protein isolated from the cells, e.g., for theproduction of antibodies and fusion proteins. In some embodiments, thecultured cells comprising a ceDNA vector for expression of FVIII proteinas disclosed herein can be used for commercial production of antibodiesor fusion proteins, e.g., serving as a cell source for small or largescale biomanufacturing of antibodies or fusion proteins. In alternativeembodiments, a ceDNA vector for expression of FVIII protein as disclosedherein is introduced into cells in a host non-human subject, for in vivoproduction of antibodies or fusion proteins, including small scaleproduction as well as for commercial large scale FVIII proteinproduction.

The ceDNA vectors for expression of FVIII protein as disclosed hereincan be used in both veterinary and medical applications. Suitablesubjects for ex vivo gene delivery methods as described above includeboth avians (e.g., chickens, ducks, geese, quail, turkeys and pheasants)and mammals (e.g., humans, bovines, ovines, caprines, equines, felines,canines, and lagomorphs), with mammals being preferred. Human subjectsare most preferred. Human subjects include neonates, infants, juveniles,and adults.

D. Dose Ranges

Provided herein are methods of treatment comprising administering to thesubject an effective amount of a composition comprising a ceDNA vectorencoding an FVIII protein as described herein. As will be appreciated bya skilled practitioner, the term “effective amount” refers to the amountof the ceDNA composition administered that results in expression of theFVIII protein in a “therapeutically effective amount” for the treatmentof hemophilia A.

In vivo and/or in vitro assays can optionally be employed to helpidentify optimal dosage ranges for use. The precise dose to be employedin the formulation will also depend on the route of administration, andthe seriousness of the condition, and should be decided according to thejudgment of the person of ordinary skill in the art and each subject'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

A ceDNA vectors for expression of FVIII protein as disclosed herein isadministered in sufficient amounts to transfect the cells of a desiredtissue and to provide sufficient levels of gene transfer and expressionwithout undue adverse effects. Conventional and pharmaceuticallyacceptable routes of administration include, but are not limited to,those described above in the “Administration” section, such as directdelivery to the selected organ (e.g., intraportal delivery to theliver), oral, inhalation (including intranasal and intratrachealdelivery), intraocular, intravenous, intramuscular, subcutaneous,intradermal, intratumoral, and other parental routes of administration.Routes of administration can be combined, if desired.

The dose of the amount of a ceDNA vectors for expression of FVIIIprotein as disclosed herein required to achieve a particular“therapeutic effect,” will vary based on several factors including, butnot limited to: the route of nucleic acid administration, the level ofgene or RNA expression required to achieve a therapeutic effect, thespecific disease or disorder being treated, and the stability of thegene(s), RNA product(s), or resulting expressed protein(s). One of skillin the art can readily determine a ceDNA vector dose range to treat apatient having a particular disease or disorder based on theaforementioned factors, as well as other factors that are well known inthe art.

Dosage regime can be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide can be repeatedlyadministered, e.g., several doses can be administered daily, or the dosecan be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

A “therapeutically effective dose” will fall in a relatively broad rangethat can be determined through clinical trials and will depend on theparticular application (neural cells will require very small amounts,while systemic injection would require large amounts). For example, fordirect in vivo injection into skeletal or cardiac muscle of a humansubject, a therapeutically effective dose will be on the order of fromabout 1 μg to 100 g of the ceDNA vector. If exosomes or microparticlesare used to deliver the ceDNA vector, then a therapeutically effectivedose can be determined experimentally, but is expected to deliver from 1μg to about 100 g of vector. Moreover, a therapeutically effective doseis an amount ceDNA vector that expresses a sufficient amount of thetransgene to have an effect on the subject that results in a reductionin one or more symptoms of the disease, but does not result insignificant off-target or significant adverse side effects. In oneembodiment, a “therapeutically effective amount” is an amount of anexpressed FVIII protein that is sufficient to produce a statisticallysignificant, measurable change in expression of hemophilia A biomarkeror reduction of a given disease symptom. Such effective amounts can begauged in clinical trials as well as animal studies for a given ceDNAvector composition.

Formulation of pharmaceutically acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

For in vitro transfection, an effective amount of a ceDNA vectors forexpression of FVIII protein as disclosed herein to be delivered to cells(1×10⁶ cells) will be on the order of 0.1 to 100 μg ceDNA vector,preferably 1 to 20 μg, and more preferably 1 to 15 μg or 8 to 10 μg.Larger ceDNA vectors will require higher doses. If exosomes ormicroparticles are used, an effective in vitro dose can be determinedexperimentally but would be intended to deliver generally the sameamount of the ceDNA vector.

For the treatment of hemophilia A, the appropriate dosage of a ceDNAvector that expresses an FVIII protein as disclosed herein will dependon the specific type of disease to be treated, the type of a FVIIIprotein, the severity and course of the hemophilia A disease, previoustherapy, the patient's clinical history and response to the antibody,and the discretion of the attending physician. The ceDNA vector encodinga FVIII protein is suitably administered to the patient at one time orover a series of treatments. Various dosing schedules including, but notlimited to, single or multiple administrations over various time-points,bolus administration, and pulse infusion are contemplated herein.

Depending on the type and severity of the disease, a ceDNA vector isadministered in an amount that the encoded FVIII protein is expressed atabout 0.3 mg/kg to 100 mg/kg (e.g. 15 mg/kg-100 mg/kg, or any dosagewithin that range), by one or more separate administrations, or bycontinuous infusion. One typical daily dosage of the ceDNA vector issufficient to result in the expression of the encoded FVIII protein at arange from about 15 mg/kg to 100 mg/kg or more, depending on the factorsmentioned above. One exemplary dose of the ceDNA vector is an amountsufficient to result in the expression of the encoded FVIII protein asdisclosed herein in a range from about 10 mg/kg to about 50 mg/kg. Thus,one or more doses of a ceDNA vector in an amount sufficient to result inthe expression of the encoded FVIII protein at about 0.5 mg/kg, 1 mg/kg,1.5 mg/kg, 2.0 mg/kg, 3 mg/kg, 4.0 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg,20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg (or any combination thereof) maybe administered to the patient. In some embodiments, the ceDNA vector isan amount sufficient to result in the expression of the encoded FVIIIprotein for a total dose in the range of 50 mg to 2500 mg. An exemplarydose of a ceDNA vector is an amount sufficient to result in the totalexpression of the encoded FVIII protein at about 50 mg, about 100 mg,200 mg, 300 mg, 400 mg, about 500 mg, about 600 mg, about 700 mg, about720 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1200 mg,about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2050 mg, about2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, or about 2500 mg(or any combination thereof). As the expression of the FVIII proteinfrom ceDNA vector can be carefully controlled by regulatory switchesherein, or alternatively multiple dose of the ceDNA vector administeredto the subject, the expression of the FVIII protein from the ceDNAvector can be controlled in such a way that the doses of the expressedFVIII protein may be administered intermittently, e.g. every week, everytwo weeks, every three weeks, every four weeks, every month, every twomonths, every three months, or every six months from the ceDNA vector.The progress of this therapy can be monitored by conventional techniquesand assays.

In certain embodiments, a ceDNA vector is administered an amountsufficient to result in the expression of the encoded FVIII protein at adose of 15 mg/kg, 30 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg or aflat dose, e.g., 300 mg, 500 mg, 700 mg, 800 mg, or higher. In someembodiments, the expression of the FVIII protein from the ceDNA vectoris controlled such that the FVIII protein is expressed every day, everyother day, every week, every 2 weeks or every 4 weeks for a period oftime. In some embodiments, the expression of the FVIII protein from theceDNA vector is controlled such that the FVIII protein is expressedevery 2 weeks or every 4 weeks for a period of time. In certainembodiments, the period of time is 6 months, one year, eighteen months,two years, five years, ten years, 15 years, 20 years, or the lifetime ofthe patient.

Treatment can involve administration of a single dose or multiple doses.In some embodiments, more than one dose can be administered to asubject; in fact, multiple doses can be administered as needed, becausethe ceDNA vector elicits does not elicit an anti-capsid host immuneresponse due to the absence of a viral capsid. As such, one of skill inthe art can readily determine an appropriate number of doses. The numberof doses administered can, for example, be on the order of 1-100,preferably 2-20 doses.

Without wishing to be bound by any particular theory, the lack oftypical anti-viral immune response elicited by administration of a ceDNAvector as described by the disclosure (i.e., the absence of capsidcomponents) allows the ceDNA vector for expression of FVIII protein tobe administered to a host on multiple occasions. In some embodiments,the number of occasions in which a heterologous nucleic acid isdelivered to a subject is in a range of 2 to 10 times (e.g., 2, 3, 4, 5,6, 7, 8, 9, or 10 times). In some embodiments, a ceDNA vector isdelivered to a subject more than 10 times.

In some embodiments, a dose of a ceDNA vector for expression of FVIIIprotein as disclosed herein is administered to a subject no more thanonce per calendar day (e.g., a 24-hour period). In some embodiments, adose of a ceDNA vector is administered to a subject no more than onceper 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of aceDNA vector for expression of FVIII protein as disclosed herein isadministered to a subject no more than once per calendar week (e.g., 7calendar days). In some embodiments, a dose of a ceDNA vector isadministered to a subject no more than bi-weekly (e.g., once in a twocalendar week period). In some embodiments, a dose of a ceDNA vector isadministered to a subject no more than once per calendar month (e.g.,once in 30 calendar days). In some embodiments, a dose of a ceDNA vectoris administered to a subject no more than once per six calendar months.In some embodiments, a dose of a ceDNA vector is administered to asubject no more than once per calendar year (e.g., 365 days or 366 daysin a leap year).

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) of a ceDNA vector for expression ofFVIII protein as disclosed herein may be employed to achieve the desiredlevel of gene expression over a period of various intervals, e.g.,daily, weekly, monthly, yearly, etc.

In some embodiments, a therapeutic a FVIII protein encoded by a ceDNAvector as disclosed herein can be regulated by a regulatory switch,inducible or repressible promotor so that it is expressed in a subjectfor at least 1 hour, at least 2 hours, at least 5 hours, at least 10hours, at least 12 hours, at least 18 hours, at least 24 hours, at least36 hours, at least 48 hours, at least 72 hours, at least 1 week, atleast 2 weeks, at least 1 month, at least 2 months, at least 6 months,at least 12 months/one year, at least 2 years, at least 5 years, atleast 10 years, at least 15 years, at least 20 years, at least 30 years,at least 40 years, at least 50 years or more. In one embodiment, theexpression can be achieved by repeated administration of the ceDNAvectors described herein at predetermined or desired intervals.Alternatively, a ceDNA vector for expression of FVIII protein asdisclosed herein can further comprise components of a gene editingsystem (e.g., CRISPR/Cas, TALENs, zinc finger endonucleases etc.) topermit insertion of the one or more nucleic acid sequences encoding theFVIII protein for substantially permanent treatment or “curing” thedisease. Such ceDNA vectors comprising gene editing components aredisclosed in International Application PCT/US18/64242, and can includethe 5′ and 3′ homology arms (e.g., SEQ ID NO: 151-154, or sequences withat least 40%, 50%, 60%, 70% or 80% homology thereto) for insertion ofthe nucleic acid encoding the a FVIII protein into safe harbor regions,such as, but not including albumin gene or CCR5 gene. By way of example,a ceDNA vector expressing a FVIII protein can comprise at least onegenomic safe harbor (GSH)-specific homology arms for insertion of theFVIII transgene into a genomic safe harbor is disclosed in InternationalPatent Application PCT/US2019/020225, filed on Mar. 1, 2019, which isincorporated herein in its entirety by reference.

The duration of treatment depends upon the subject's clinical progressand responsiveness to therapy. Continuous, relatively low maintenancedoses are contemplated after an initial higher therapeutic dose.

E. Unit Dosage Forms

In some embodiments, the pharmaceutical compositions comprising a ceDNAvector for expression of FVIII protein as disclosed herein canconveniently be presented in unit dosage form. A unit dosage form willtypically be adapted to one or more specific routes of administration ofthe pharmaceutical composition. In some embodiments, the unit dosageform is adapted for droplets to be administered directly to the eye. Insome embodiments, the unit dosage form is adapted for administration byinhalation. In some embodiments, the unit dosage form is adapted foradministration by a vaporizer. In some embodiments, the unit dosage formis adapted for administration by a nebulizer. In some embodiments, theunit dosage form is adapted for administration by an aerosolizer. Insome embodiments, the unit dosage form is adapted for oraladministration, for buccal administration, or for sublingualadministration. In some embodiments, the unit dosage form is adapted forintravenous, intramuscular, or subcutaneous administration. In someembodiments, the unit dosage form is adapted for subretinal injection,suprachoroidal injection or intravitreal injection.

In some embodiments, the unit dosage form is adapted for intrathecal orintracerebroventricular administration. In some embodiments, thepharmaceutical composition is formulated for topical administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect.

X. Methods of Treatment

The technology described herein also demonstrates methods for making, aswell as methods of using the disclosed ceDNA vectors for expression ofFVIII protein in a variety of ways, including, for example, ex vivo, exsitu, in vitro and in vivo applications, methodologies, diagnosticprocedures, and/or gene therapy regimens.

In one embodiment, the expressed therapeutic FVIII protein expressedfrom a ceDNA vector as disclosed herein is functional for the treatmentof disease. In a preferred embodiment, the therapeutic FVIII proteindoes not cause an immune system reaction, unless so desired.

Provided herein is a method of treating hemophilia A in a subjectcomprising introducing into a target cell in need thereof (for example,a muscle cell or tissue, or other affected cell type) of the subject atherapeutically effective amount of a ceDNA vector for expression ofFVIII protein as disclosed herein, optionally with a pharmaceuticallyacceptable carrier. While the ceDNA vector can be introduced in thepresence of a carrier, such a carrier is not required. The ceDNA vectorimplemented comprises a nucleotide sequence encoding an FVIII protein asdescribed herein useful for treating the disease. In particular, a ceDNAvector for expression of FVIII protein as disclosed herein may comprisea desired FVIII protein DNA sequence operably linked to control elementscapable of directing transcription of the desired FVIII protein encodedby the exogenous DNA sequence when introduced into the subject. TheceDNA vector for expression of FVIII protein as disclosed herein can beadministered via any suitable route as provided above, and elsewhereherein.

Disclosed herein are ceDNA vector compositions and formulations forexpression of FVIII protein as disclosed herein that include one or moreof the ceDNA vectors of the present invention together with one or morepharmaceutically-acceptable buffers, diluents, or excipients. Suchcompositions may be included in one or more diagnostic or therapeutickits, for diagnosing, preventing, treating or ameliorating one or moresymptoms of hemophilia A. In one aspect the disease, injury, disorder,trauma or dysfunction is a human disease, injury, disorder, trauma ordysfunction.

Another aspect of the technology described herein provides a method forproviding a subject in need thereof with a diagnostically- ortherapeutically-effective amount of a ceDNA vector for expression ofFVIII protein as disclosed herein, the method comprising providing to acell, tissue or organ of a subject in need thereof, an amount of theceDNA vector as disclosed herein; and for a time effective to enableexpression of the FVIII protein from the ceDNA vector thereby providingthe subject with a diagnostically- or a therapeutically-effective amountof the FVIII protein expressed by the ceDNA vector. In a further aspect,the subject is human.

Another aspect of the technology described herein provides a method fordiagnosing, preventing, treating, or ameliorating at least one or moresymptoms of hemophilia A, a disorder, a dysfunction, an injury, anabnormal condition, or trauma in a subject. In an overall and generalsense, the method includes at least the step of administering to asubject in need thereof one or more of the disclosed ceDNA vector forFVIII protein production, in an amount and for a time sufficient todiagnose, prevent, treat or ameliorate the one or more symptoms of thedisease, disorder, dysfunction, injury, abnormal condition, or trauma inthe subject. In such an embodiment, the subject can be evaluated forefficacy of the FVIII protein, or alternatively, detection of the FVIIIprotein or tissue location (including cellular and subcellular location)of the FVIII protein in the subject. As such, the ceDNA vector forexpression of FVIII protein as disclosed herein can be used as an invivo diagnostic tool, e.g., for the detection of cancer or otherindications. In a further aspect, the subject is human.

Another aspect is use of a ceDNA vector for expression of FVIII proteinas disclosed herein as a tool for treating or reducing one or moresymptoms of hemophilia A or disease states. There are a number ofinherited diseases in which defective genes are known, and typicallyfall into two classes: deficiency states, usually of enzymes, which aregenerally inherited in a recessive manner, and unbalanced states, whichmay involve regulatory or structural proteins, and which are typicallybut not always inherited in a dominant manner For unbalanced diseasestates, a ceDNA vector for expression of FVIII protein as disclosedherein can be used to create hemophilia A state in a model system, whichcould then be used in efforts to counteract the disease state. Thus, theceDNA vector for expression of FVIII protein as disclosed herein permitthe treatment of genetic diseases. As used herein, hemophilia A state istreated by partially or wholly remedying the deficiency or imbalancethat causes the disease or makes it more severe.

A. Host Cells

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein delivers the FVIII protein transgene into a subjecthost cell. In some embodiments, the cells are photoreceptor cells. Insome embodiments, the cells are RPE cells. In some embodiments, thesubject host cell is a human host cell, including, for example bloodcells, stem cells, hematopoietic cells, CD34⁺ cells, liver cells, cancercells, vascular cells, muscle cells, pancreatic cells, neural cells,ocular or retinal cells, epithelial or endothelial cells, dendriticcells, fibroblasts, or any other cell of mammalian origin, including,without limitation, hepatic (i.e., liver) cells, lung cells, cardiaccells, pancreatic cells, intestinal cells, diaphragmatic cells, renal(i.e., kidney) cells, neural cells, blood cells, bone marrow cells, orany one or more selected tissues of a subject for which gene therapy iscontemplated. In one aspect, the subject host cell is a human host cell.

The present disclosure also relates to recombinant host cells asmentioned above, including a ceDNA vector for expression of FVIIIprotein as disclosed herein. Thus, one can use multiple host cellsdepending on the purpose as is obvious to the skilled artisan. Aconstruct or a ceDNA vector for expression of FVIII protein as disclosedherein including donor sequence is introduced into a host cell so thatthe donor sequence is maintained as a chromosomal integrant as describedearlier. The term host cell encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The choice of a host cell will to a large extentdepend upon the donor sequence and its source.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell. In one embodiment, the host cell is a human cell(e.g., a primary cell, a stem cell, or an immortalized cell line). Insome embodiments, the host cell can be administered a ceDNA vector forexpression of FVIII protein as disclosed herein ex vivo and thendelivered to the subject after the gene therapy event. A host cell canbe any cell type, e.g., a somatic cell or a stem cell, an inducedpluripotent stem cell, or a blood cell, e.g., T-cell or B-cell, or bonemarrow cell. In certain embodiments, the host cell is an allogenic cell.For example, T-cell genome engineering is useful for cancerimmunotherapies, disease modulation such as HIV therapy (e.g., receptorknock out, such as CXCR4 and CCR5) and immunodeficiency therapies. MHCreceptors on B-cells can be targeted for immunotherapy. In someembodiments, gene modified host cells, e.g., bone marrow stem cells,e.g., CD34⁺ cells, or induced pluripotent stem cells can be transplantedback into a patient for expression of a therapeutic protein.

B. Additional Diseases for Gene Therapy:

In general, a ceDNA vector for expression of FVIII protein as disclosedherein can be used to deliver any FVIII protein in accordance with thedescription above to treat, prevent, or ameliorate the symptomsassociated with hemophilia A related to an aberrant protein expressionor gene expression in a subject.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein can be used to deliver an FVIII protein to skeletal,cardiac or diaphragm muscle, for production of an FVIII protein forsecretion and circulation in the blood or for systemic delivery to othertissues to treat, ameliorate, and/or prevent hemophilia A.

The a ceDNA vector for expression of FVIII protein as disclosed hereincan be administered to the lungs of a subject by any suitable means,optionally by administering an aerosol suspension of respirableparticles comprising the ceDNA vectors, which the subject inhales. Therespirable particles can be liquid or solid. Aerosols of liquidparticles comprising the ceDNA vectors may be produced by any suitablemeans, such as with a pressure-driven aerosol nebulizer or an ultrasonicnebulizer, as is known to those of skill in the art. See, e.g., U.S.Pat. No. 4,501,729. Aerosols of solid particles comprising the ceDNAvectors may likewise be produced with any solid particulate medicamentaerosol generator, by techniques known in the pharmaceutical art.

In some embodiments, a ceDNA vector for expression of FVIII protein asdisclosed herein can be administered to tissues of the CNS (e.g., brain,eye).

Ocular disorders that may be treated, ameliorated, or prevented with aceDNA vector for expression of FVIII protein as disclosed herein includeophthalmic disorders involving the retina, posterior tract, and opticnerve (e.g., retinitis pigmentosa, diabetic retinopathy and otherretinal degenerative diseases, uveitis, age-related maculardegeneration, glaucoma). Many ophthalmic diseases and disorders areassociated with one or more of three types of indications: (1)angiogenesis, (2) inflammation, and (3) degeneration. In someembodiments, the ceDNA vector as disclosed herein can be employed todeliver anti-angiogenic factors; anti-inflammatory factors; factors thatretard cell degeneration, promote cell sparing, or promote cell growthand combinations of the foregoing. Diabetic retinopathy, for example, ischaracterized by angiogenesis. Diabetic retinopathy can be treated bydelivering one or more anti-angiogenic antibodies or fusion proteinseither intraocularly (e.g., in the vitreous) or periocularly (e.g., inthe sub-Tenon's region). Additional ocular diseases that may be treated,ameliorated, or prevented with the ceDNA vectors of the inventioninclude geographic atrophy, vascular or “wet” macular degeneration, PKU,Leber Congenital Amaurosis (LCA), Usher syndrome, pseudoxanthomaelasticum (PXE), x-linked retinitis pigmentosa (XLRP), x-linkedretinoschisis (XLRS), Choroideremia, Leber hereditary optic neuropathy(LHON), Archomatopsia, cone-rod dystrophy, Fuchs endothelial cornealdystrophy, diabetic macular edema and ocular cancer and tumors.

In some embodiments, inflammatory ocular diseases or disorders (e.g.,uveitis) can be treated, ameliorated, or prevented by a ceDNA vector forexpression of FVIII protein as disclosed herein. One or moreanti-inflammatory antibodies or fusion proteins can be expressed byintraocular (e.g., vitreous or anterior chamber) administration of theceDNA vector as disclosed herein. In some embodiments, a ceDNA vectorfor expression of FVIII protein as disclosed herein can encode an FVIIIprotein that is associated with transgene encoding a reporterpolypeptide (e.g., an enzyme such as Green Fluorescent Protein, oralkaline phosphatase). In some embodiments, a transgene that encodes areporter protein useful for experimental or diagnostic purposes, isselected from any of: β-lactamase, β-galactosidase (LacZ), alkalinephosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art. In some aspects, ceDNA vectors expressing an FVIIIprotein linked to a reporter polypeptide may be used for diagnosticpurposes, as well as to determine efficacy or as markers of the ceDNAvector's activity in the subject to which they are administered.

C. Testing for Successful Gene Expression Using a ceDNA Vector

Assays well known in the art can be used to test the efficiency of genedelivery of an FVIII protein by a ceDNA vector can be performed in bothin vitro and in vivo models. Levels of the expression of the FVIIIprotein by ceDNA can be assessed by one skilled in the art by measuringmRNA and protein levels of the FVIII protein (e.g., reversetranscription PCR, western blot analysis, and enzyme-linkedimmunosorbent assay (ELISA)). In one embodiment, ceDNA comprises areporter protein that can be used to assess the expression of the FVIIIprotein, for example by examining the expression of the reporter proteinby fluorescence microscopy or a luminescence plate reader. For in vivoapplications, protein function assays can be used to test thefunctionality of a given FVIII protein to determine if gene expressionhas successfully occurred. One skilled will be able to determine thebest test for measuring functionality of an FVIII protein expressed bythe ceDNA vector in vitro or in vivo.

It is contemplated herein that the effects of gene expression of anFVIII protein from the ceDNA vector in a cell or subject can last for atleast 1 month, at least 2 months, at least 3 months, at least fourmonths, at least 5 months, at least six months, at least 10 months, atleast 12 months, at least 18 months, at least 2 years, at least 5 years,at least 10 years, at least 20 years, or can be permanent.

In some embodiments, an FVIII protein in the expression cassette,expression construct, or ceDNA vector described herein can be codonoptimized for the host cell. As used herein, the term “codon optimized”or “codon optimization” refers to the process of modifying a nucleicacid sequence for enhanced expression in the cells of the vertebrate ofinterest, e.g., mouse or human (e.g., humanized), by replacing at leastone, more than one, or a significant number of codons of the nativesequence (e.g., a prokaryotic sequence) with codons that are morefrequently or most frequently used in the genes of that vertebrate.Various species exhibit particular bias for certain codons of aparticular amino acid. Typically, codon optimization does not alter theamino acid sequence of the original translated protein. Optimized codonscan be determined using e.g., Aptagen's Gene Forge® codon optimizationand custom gene synthesis platform (Aptagen, Inc.) or another publiclyavailable database.

D. Determining Efficacy by Assessing FVIII Protein Expression from theceDNA Vector

Essentially any method known in the art for determining proteinexpression can be used to analyze expression of a FVIII protein from aceDNA vector. Non-limiting examples of such methods/assays includeenzyme-linked immunoassay (ELISA), affinity ELISA, ELISPOT, serialdilution, flow cytometry, surface plasmon resonance analysis, kineticexclusion assay, mass spectrometry, Western blot, immunoprecipitation,and PCR.

For assessing FVIII protein expression in vivo, a biological sample canbe obtained from a subject for analysis. Exemplary biological samplesinclude a biofluid sample, a body fluid sample, blood (including wholeblood), serum, plasma, urine, saliva, a biopsy and/or tissue sample etc.A biological sample or tissue sample can also refer to a sample oftissue or fluid isolated from an individual including, but not limitedto, tumor biopsy, stool, spinal fluid, pleural fluid, nipple aspirates,lymph fluid, the external sections of the skin, respiratory, intestinal,and genitourinary tracts, tears, saliva, breast milk, cells (including,but not limited to, blood cells), tumors, organs, and also samples of invitro cell culture constituent. The term also includes a mixture of theabove-mentioned samples. The term “sample” also includes untreated orpretreated (or pre-processed) biological samples. In some embodiments,the sample used for the assays and methods described herein comprises aserum sample collected from a subject to be tested.

E. Determining Efficacy of the Expressed FVIII Protein by ClinicalParameters

The efficacy of a given FVIII protein expressed by a ceDNA vector forhemophilia A (i.e., functional expression) can be determined by theskilled clinician. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of hemophilia A is/are altered in a beneficial manner, orother clinically accepted symptoms or markers of disease are improved,or ameliorated, e.g., by at least 10% following treatment with a ceDNAvector encoding a therapeutic FVIII protein as described herein.Efficacy can also be measured by failure of an individual to worsen asassessed by stabilization of hemophilia A, or the need for medicalinterventions (i.e., progression of the disease is halted or at leastslowed). Methods of measuring these indicators are known to those ofskill in the art and/or described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human, or a mammal) and includes: (1) inhibitinghemophilia A, e.g., arresting, or slowing progression of hemophilia A;or (2) relieving the hemophilia A, e.g., causing regression of ahemophilia A symptom; and (3) preventing or reducing the likelihood ofthe development of the hemophilia A disease, or preventing secondarydiseases/disorders associated with hemophilia A. An effective amount forthe treatment of a disease means that amount which, when administered toa mammal in need thereof, is sufficient to result in effective treatmentas that term is defined herein, for that disease. Efficacy of an agentcan be determined by assessing physical indicators that are particularto hemophilia A disease. A physician can assess for any one or more ofclinical symptoms of hemophilia A which include: unexplained andexcessive bleeding from cuts or injuries, or after surgery or dentalwork; many large or deep bruises; unusual bleeding after vaccinations;pain, swelling or tightness in your joints; blood in your urine orstool; nosebleeds without a known cause; in infants, unexplainedirritability.

XI. Various Applications of ceDNA Vectors Expressing Antibodies orFusion Proteins

As disclosed herein, the compositions and ceDNA vectors for expressionof FVIII protein as described herein can be used to express an FVIIIprotein for a range of purposes. In one embodiment, the ceDNA vectorexpressing an FVIII protein can be used to create a somatic transgenicanimal model harboring the transgene, e.g., to study the function ordisease progression of hemophilia A. In some embodiments, a ceDNA vectorexpressing an FVIII protein is useful for the treatment, prevention, oramelioration of hemophilia A states or disorders in a mammalian subject.

In some embodiments the FVIII protein can be expressed from the ceDNAvector in a subject in a sufficient amount to treat a disease associatedwith increased expression, increased activity of the gene product, orinappropriate upregulation of a gene.

In some embodiments the FVIII protein can be expressed from the ceDNAvector in a subject in a sufficient amount to treat hemophilia A with areduced expression, lack of expression or dysfunction of a protein.

It will be appreciated by one of ordinary skill in the art that thetransgene may not be an open reading frame of a gene to be transcribeditself; instead it may be a promoter region or repressor region of atarget gene, and the ceDNA vector may modify such region with theoutcome of so modulating the expression of the FVIII gene.

The compositions and ceDNA vectors for expression of FVIII protein asdisclosed herein can be used to deliver an FVIII protein for variouspurposes as described above.

In some embodiments, the transgene encodes one or more FVIII proteinswhich are useful for the treatment, amelioration, or prevention ofhemophilia A states in a mammalian subject. The FVIII protein expressedby the ceDNA vector is administered to a patient in a sufficient amountto treat hemophilia A associated with an abnormal gene sequence, whichcan result in any one or more of the following: increased proteinexpression, over activity of the protein, reduced expression, lack ofexpression or dysfunction of the target gene or protein.

In some embodiments, the ceDNA vectors for expression of FVIII proteinas disclosed herein are envisioned for use in diagnostic and screeningmethods, whereby an FVIII protein is transiently or stably expressed ina cell culture system, or alternatively, a transgenic animal model.

Another aspect of the technology described herein provides a method oftransducing a population of mammalian cells with a ceDNA vector forexpression of FVIII protein as disclosed herein. In an overall andgeneral sense, the method includes at least the step of introducing intoone or more cells of the population, a composition that comprises aneffective amount of one or more of the ceDNA vectors for expression ofFVIII protein as disclosed herein.

Additionally, the present invention provides compositions, as well astherapeutic and/or diagnostic kits that include one or more of thedisclosed ceDNA vectors for expression of FVIII protein as disclosedherein or ceDNA compositions, formulated with one or more additionalingredients, or prepared with one or more instructions for their use.

A cell to be administered a ceDNA vector for expression of FVIII proteinas disclosed herein may be of any type, including but not limited toneural cells (including cells of the peripheral and central nervoussystems, in particular, brain cells), lung cells, retinal cells,epithelial cells (e.g., gut and respiratory epithelial cells), musclecells, dendritic cells, pancreatic cells (including islet cells),hepatic cells, myocardial cells, bone cells (e.g., bone marrow stemcells), hematopoietic stem cells, spleen cells, keratinocytes,fibroblasts, endothelial cells, prostate cells, germ cells, and thelike. Alternatively, the cell may be any progenitor cell. As a furtheralternative, the cell can be a stem cell (e.g., neural stem cell, liverstem cell). As still a further alternative, the cell may be a cancer ortumor cell. Moreover, the cells can be from any species of origin, asindicated above.

A. Production and Purification of ceDNA Vectors Expressing FVIII

The ceDNA vectors disclosed herein are to be used to produce FVIIIprotein either in vitro or in vivo. The FVIII proteins produced in thismanner can be isolated, tested for a desired function, and purified forfurther use in research or as a therapeutic treatment. Each system ofprotein production has its own advantages/disadvantages. While proteinsproduced in vitro can be easily purified and can proteins in a shorttime, proteins produced in vivo can have post-translationalmodifications, such as glycosylation.

FVIII therapeutic protein produced using ceDNA vectors can be purifiedusing any method known to those of skill in the art, for example, ionexchange chromatography, affinity chromatography, precipitation, orelectrophoresis.

An FVIII therapeutic protein produced by the methods and compositionsdescribed herein can be tested for binding to the desired targetprotein.

EXAMPLES

The following examples are provided by way of illustration notlimitation. It will be appreciated by one of ordinary skill in the artthat ceDNA vectors can be constructed from any of the wild-type ormodified ITRs described herein, and that the following exemplary methodscan be used to construct and assess the activity of such ceDNA vectors.While the methods are exemplified with certain ceDNA vectors, they areapplicable to any ceDNA vector in keeping with the description.

Example 1: Constructing ceDNA Vectors Using an Insect Cell-Based Method

Production of the ceDNA vectors using a polynucleotide constructtemplate is described in Example 1 of PCT/US18/49996, which isincorporated herein in its entirety by reference. For example, apolynucleotide construct template used for generating the ceDNA vectorsof the present invention can be a ceDNA-plasmid, a ceDNA-Bacmid, and/ora ceDNA-baculovirus. Without being limited to theory, in a permissivehost cell, in the presence of e.g., Rep, the polynucleotide constructtemplate having two symmetric ITRs and an expression construct, where atleast one of the ITRs is modified relative to a wild-type ITR sequence,replicates to produce ceDNA vectors. ceDNA vector production undergoestwo steps: first, excision (“rescue”) of template from the templatebackbone (e.g. ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genomeetc.) via Rep proteins, and second, Rep mediated replication of theexcised ceDNA vector.

An exemplary method to produce ceDNA vectors is from a ceDNA-plasmid asdescribed herein. Referring to FIGS. 1A and 1B, the polynucleotideconstruct template of each of the ceDNA-plasmids includes both a leftmodified ITR and a right modified ITR with the following between the ITRsequences: (i) an enhancer/promoter; (ii) a cloning site for atransgene; (iii) a posttranscriptional response element (e.g. thewoodchuck hepatitis virus posttranscriptional regulatory element(WPRE)); and (iv) a poly-adenylation signal (e.g. from bovine growthhormone gene (BGHpA). Unique restriction endonuclease recognition sites(R1-R6) (shown in FIG. 1A and FIG. 1B) were also introduced between eachcomponent to facilitate the introduction of new genetic components intothe specific sites in the construct. R3 (PmeI) GTTTAAAC (SEQ ID NO: 123)and R4 (PacI) TTAATTAA (SEQ ID NO: 124) enzyme sites are engineered intothe cloning site to introduce an open reading frame of a transgene.These sequences were cloned into a pFastBac HT B plasmid obtained fromThermoFisher Scientific.

Production of ceDNA-Bacmids:

DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ Competent Cells,Thermo Fisher) were transformed with either test or control plasmidsfollowing a protocol according to the manufacturer's instructions.Recombination between the plasmid and a baculovirus shuttle vector inthe DH10Bac cells were induced to generate recombinant ceDNA-bacmids.The recombinant bacmids were selected by screening a positive selectionbased on blue-white screening in E. coli (Φ80dlacZΔM15 marker providesa-complementation of the β-galactosidase gene from the bacmid vector) ona bacterial agar plate containing X-gal and IPTG with antibiotics toselect for transformants and maintenance of the bacmid and transposaseplasmids. White colonies caused by transposition that disrupts theβ-galactoside indicator gene were picked and cultured in 10 ml of media.

The recombinant ceDNA-bacmids were isolated from the E. coli andtransfected into Sf9 or Sf21 insect cells using FugeneHD to produceinfectious baculovirus. The adherent Sf9 or Sf21 insect cells werecultured in 50 ml of media in T25 flasks at 25° C. Four days later,culture medium (containing the P0 virus) was removed from the cells,filtered through a 0.45 μm filter, separating the infectious baculovirusparticles from cells or cell debris.

Optionally, the first generation of the baculovirus (P0) was amplifiedby infecting naïve Sf9 or Sf21 insect cells in 50 to 500 ml of media.Cells were maintained in suspension cultures in an orbital shakerincubator at 130 rpm at 25° C., monitoring cell diameter and viability,until cells reach a diameter of 18-19 nm (from a naïve diameter of 14-15nm), and a density of ˜4.0E+6 cells/mL. Between 3 and 8 dayspost-infection, the P1 baculovirus particles in the medium werecollected following centrifugation to remove cells and debris thenfiltration through a 0.45 μm filter.

The ceDNA-baculovirus comprising the test constructs were collected andthe infectious activity, or titer, of the baculovirus was determined.Specifically, four×20 ml Sf9 cell cultures at 2.5E+6 cells/ml weretreated with P1 baculovirus at the following dilutions: 1/1000,1/10,000, 1/50,000, 1/100,000, and incubated at 25-27° C. Infectivitywas determined by the rate of cell diameter increase and cell cyclearrest, and change in cell viability every day for 4 to 5 days.

A “Rep-plasmid” as disclosed in FIG. 8A of PCT/US18/49996, which isincorporated herein in its entirety by reference, was produced in apFASTBAC™-Dual expression vector (ThermoFisher) comprising both theRep78 (SEQ ID NO: 131 or 133) and Rep52 (SEQ ID NO: 132) or Rep68 (SEQID NO: 130) and Rep40 (SEQ ID NO: 129). The Rep-plasmid was transformedinto the DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ CompetentCells (Thermo Fisher) following a protocol provided by the manufacturer.Recombination between the Rep-plasmid and a baculovirus shuttle vectorin the DH10Bac cells were induced to generate recombinant bacmids(“Rep-bacmids”). The recombinant bacmids were selected by a positiveselection that included-blue-white screening in E. coli (Φ80dlacZΔM15marker provides α-complementation of the β-galactosidase gene from thebacmid vector) on a bacterial agar plate containing X-gal and IPTG.Isolated white colonies were picked and inoculated in 10 ml of selectionmedia (kanamycin, gentamicin, tetracycline in LB broth). The recombinantbacmids (Rep-bacmids) were isolated from the E. coli and the Rep-bacmidswere transfected into Sf9 or Sf21 insect cells to produce infectiousbaculovirus.

The Sf9 or Sf21 insect cells were cultured in 50 ml of media for 4 days,and infectious recombinant baculovirus (“Rep-baculovirus”) were isolatedfrom the culture. Optionally, the first generation Rep-baculovirus (P0)were amplified by infecting naïve Sf9 or Sf21 insect cells and culturedin 50 to 500 ml of media. Between 3 and 8 days post-infection, the P1baculovirus particles in the medium were collected either by separatingcells by centrifugation or filtration or another fractionation process.The Rep-baculovirus were collected and the infectious activity of thebaculovirus was determined. Specifically, four×20 mL Sf9 cell culturesat 2.5×10⁶ cells/mL were treated with P1 baculovirus at the followingdilutions, 1/1000, 1/10,000, 1/50,000, 1/100,000, and incubated.Infectivity was determined by the rate of cell diameter increase andcell cycle arrest, and change in cell viability every day for 4 to 5days.

ceDNA Vector Generation and Characterization

With reference to FIG. 4B, Sf9 insect cell culture media containingeither (1) a sample-containing a ceDNA-bacmid or a ceDNA-baculovirus,and (2) Rep-baculovirus described above were then added to a freshculture of Sf9 cells (2.5E+6 cells/ml, 20 ml) at a ratio of 1:1000 and1:10,000, respectively. The cells were then cultured at 130 rpm at 25°C. 4-5 days after the co-infection, cell diameter and viability aredetected. When cell diameters reached 18-20 nm with a viability of˜70-80%, the cell cultures were centrifuged, the medium was removed, andthe cell pellets were collected. The cell pellets are first resuspendedin an adequate volume of aqueous medium, either water or buffer. TheceDNA vector was isolated and purified from the cells using Qiagen MIDIPLUS™ purification protocol (Qiagen, 0.2 mg of cell pellet massprocessed per column).

Yields of ceDNA vectors produced and purified from the Sf9 insect cellswere initially determined based on UV absorbance at 260 nm.

ceDNA vectors can be assessed by identified by agarose gelelectrophoresis under native or denaturing conditions as illustrated inFIG. 4D, where (a) the presence of characteristic bands migrating attwice the size on denaturing gels versus native gels after restrictionendonuclease cleavage and gel electrophoretic analysis and (b) thepresence of monomer and dimer (2×) bands on denaturing gels foruncleaved material is characteristic of the presence of ceDNA vector.

Structures of the isolated ceDNA vectors were further analyzed bydigesting the DNA obtained from co-infected Sf9 cells (as describedherein) with restriction endonucleases selected for a) the presence ofonly a single cut site within the ceDNA vectors, and b) resultingfragments that were large enough to be seen clearly when fractionated ona 0.8% denaturing agarose gel (>800 bp). As illustrated in FIGS. 4D and4E, linear DNA vectors with a non-continuous structure and ceDNA vectorwith the linear and continuous structure can be distinguished by sizesof their reaction products—for example, a DNA vector with anon-continuous structure is expected to produce 1 kb and 2 kb fragments,while a non-encapsidated vector with the continuous structure isexpected to produce 2 kb and 4 kb fragments.

Therefore, to demonstrate in a qualitative fashion that isolated ceDNAvectors are covalently closed-ended as is required by definition, thesamples were digested with a restriction endonuclease identified in thecontext of the specific DNA vector sequence as having a singlerestriction site, preferably resulting in two cleavage products ofunequal size (e.g., 1000 bp and 2000 bp). Following digestion andelectrophoresis on a denaturing gel (which separates the twocomplementary DNA strands), a linear, non-covalently closed DNA willresolve at sizes 1000 bp and 2000 bp, while a covalently closed DNA(i.e., a ceDNA vector) will resolve at 2× sizes (2000 bp and 4000 bp),as the two DNA strands are linked and are now unfolded and twice thelength (though single stranded). Furthermore, digestion of monomeric,dimeric, and n-meric forms of the DNA vectors will all resolve as thesame size fragments due to the end-to-end linking of the multimeric DNAvectors (see FIG. 4D).

As used herein, the phrase “assay for the Identification of DNA vectorsby agarose gel electrophoresis under native gel and denaturingconditions” refers to an assay to assess the close-endedness of theceDNA by performing restriction endonuclease digestion followed byelectrophoretic assessment of the digest products. One such exemplaryassay follows, though one of ordinary skill in the art will appreciatethat many art-known variations on this example are possible. Therestriction endonuclease is selected to be a single cut enzyme for theceDNA vector of interest that will generate products of approximately1/3× and 2/3× of the DNA vector length. This resolves the bands on bothnative and denaturing gels. Before denaturation, it is important toremove the buffer from the sample. The Qiagen PCR clean-up kit ordesalting “spin columns,” e.g. GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25columns are some art-known options for the endonuclease digestion. Theassay includes for example, i) digest DNA with appropriate restrictionendonuclease(s), 2) apply to e.g., a Qiagen PCR clean-up kit, elute withdistilled water, iii) adding 10× denaturing solution (10×=0.5 M NaOH, 10mM EDTA), add 10× dye, not buffered, and analyzing, together with DNAladders prepared by adding 10× denaturing solution to 4×, on a 0.8-1.0%gel previously incubated with 1 mM EDTA and 200 mM NaOH to ensure thatthe NaOH concentration is uniform in the gel and gel box, and runningthe gel in the presence of 1× denaturing solution (50 mM NaOH, 1 mMEDTA). One of ordinary skill in the art will appreciate what voltage touse to run the electrophoresis based on size and desired timing ofresults. After electrophoresis, the gels are drained and neutralized in1×TBE or TAE and transferred to distilled water or 1×TBE/TAE with 1×SYBRGold. Bands can then be visualized with e.g. Thermo Fisher, SYBR® GoldNucleic Acid Gel Stain (10,000× Concentrate in DMSO) and epifluorescentlight (blue) or UV (312 nm).

The purity of the generated ceDNA vector can be assessed using anyart-known method. As one exemplary and non-limiting method, contributionof ceDNA-plasmid to the overall UV absorbance of a sample can beestimated by comparing the fluorescent intensity of ceDNA vector to astandard. For example, if based on UV absorbance 4 μg of ceDNA vectorwas loaded on the gel, and the ceDNA vector fluorescent intensity isequivalent to a 2 kb band which is known to be 1 μg, then there is 1 μgof ceDNA vector, and the ceDNA vector is 25% of the total UV absorbingmaterial. Band intensity on the gel is then plotted against thecalculated input that band represents—for example, if the total ceDNAvector is 8 kb, and the excised comparative band is 2 kb, then the bandintensity would be plotted as 25% of the total input, which in this casewould be 0.25 μg for 1.0 μg input. Using the ceDNA vector plasmidtitration to plot a standard curve, a regression line equation is thenused to calculate the quantity of the ceDNA vector band, which can thenbe used to determine the percent of total input represented by the ceDNAvector, or percent purity.

For comparative purposes, Example 1 describes the production of ceDNAvectors using an insect cell based method and a polynucleotide constructtemplate, and is also described in Example 1 of PCT/US18/49996, which isincorporated herein in its entirety by reference. For example, apolynucleotide construct template used for generating the ceDNA vectorsof the present invention according to Example 1 can be a ceDNA-plasmid,a ceDNA-Bacmid, and/or a ceDNA-baculovirus. Without being limited totheory, in a permissive host cell, in the presence of e.g., Rep, thepolynucleotide construct template having two symmetric ITRs and anexpression construct, where at least one of the ITRs is modifiedrelative to a wild-type ITR sequence, replicates to produce ceDNAvectors. ceDNA vector production undergoes two steps: first, excision(“rescue”) of template from the template backbone (e.g. ceDNA-plasmid,ceDNA-bacmid, ceDNA-baculovirus genome etc.) via Rep proteins, andsecond, Rep mediated replication of the excised ceDNA vector.

An exemplary method to produce ceDNA vectors in a method using insectcell is from a ceDNA-plasmid as described herein. Referring to FIGS. 1Aand 1B, the polynucleotide construct template of each of theceDNA-plasmids includes both a left modified ITR and a right modifiedITR with the following between the ITR sequences: (i) anenhancer/promoter; (ii) a cloning site for a transgene; (iii) aposttranscriptional response element (e.g. the woodchuck hepatitis virusposttranscriptional regulatory element (WPRE)); and (iv) apoly-adenylation signal (e.g. from bovine growth hormone gene (BGHpA).Unique restriction endonuclease recognition sites (R1-R6) (shown in FIG.1A and FIG. 1B) were also introduced between each component tofacilitate the introduction of new genetic components into the specificsites in the construct. R3 (PmeI) GTTTAAAC (SEQ ID NO: 123) and R4 (Pad)TTAATTAA (SEQ ID NO: 124) enzyme sites are engineered into the cloningsite to introduce an open reading frame of a transgene. These sequenceswere cloned into a pFastBac HT B plasmid obtained from ThermoFisherScientific.

Example 2: Synthetic ceDNA Production Via Excision from aDouble-Stranded DNA Molecule

Synthetic production of the ceDNA vectors is described in Examples 2-6of International Application PCT/US19/14122, filed Jan. 18, 2019, whichis incorporated herein in its entirety by reference. One exemplarymethod of producing a ceDNA vector using a synthetic method thatinvolves the excision of a double-stranded DNA molecule. In brief, aceDNA vector can be generated using a double stranded DNA construct,e.g., see FIGS. 7A-8E of PCT/US19/14122. In some embodiments, the doublestranded DNA construct is a ceDNA plasmid, e.g., see, e.g., FIG. 6 inInternational patent application PCT/US2018/064242, filed Dec. 6, 2018).

In some embodiments, a construct to make a ceDNA vector comprises aregulatory switch as described herein.

For illustrative purposes, Example 2 describes producing ceDNA vectorsas exemplary closed-ended DNA vectors generated using this method.However, while ceDNA vectors are exemplified in this Example toillustrate in vitro synthetic production methods to generate aclosed-ended DNA vector by excision of a double-stranded polynucleotidecomprising the ITRs and expression cassette (e.g., heterologous nucleicacid sequence) followed by ligation of the free 3′ and 5′ ends asdescribed herein, one of ordinary skill in the art is aware that onecan, as illustrated above, modify the double stranded DNA polynucleotidemolecule such that any desired closed-ended DNA vector is generated,including but not limited to, doggybone DNA, dumbbell DNA and the like.Exemplary ceDNA vectors for production of antibodies or fusion proteinsthat can be produced by the synthetic production method described inExample 2 are discussed in the sections entitled “III ceDNA vectors ingeneral”. Exemplary antibodies and fusion proteins expressed by theceDNA vectors are described in the section entitled “IIC Exemplaryantibodies and fusion proteins expressed by the ceDNA vectors”.

The method involves (i) excising a sequence encoding the expressioncassette from a double-stranded DNA construct and (ii) forming hairpinstructures at one or more of the ITRs and (iii) joining the free 5′ and3′ ends by ligation, e.g., by T4 DNA ligase.

The double-stranded DNA construct comprises, in 5′ to 3′ order: a firstrestriction endonuclease site; an upstream ITR; an expression cassette;a downstream ITR; and a second restriction endonuclease site. Thedouble-stranded DNA construct is then contacted with one or morerestriction endonucleases to generate double-stranded breaks at both ofthe restriction endonuclease sites. One endonuclease can target bothsites, or each site can be targeted by a different endonuclease as longas the restriction sites are not present in the ceDNA vector template.This excises the sequence between the restriction endonuclease sitesfrom the rest of the double-stranded DNA construct (see FIG. 9 ofPCT/US19/14122). Upon ligation a closed-ended DNA vector is formed.

One or both of the ITRs used in the method may be wild-type ITRs.Modified ITRs may also be used, where the modification can includedeletion, insertion, or substitution of one or more nucleotides from thewild-type ITR in the sequences forming B and B′ arm and/or C and C′ arm(see, e.g., FIGS. 6-8 and 10 FIG. 11B of PCT/US19/14122), and may havetwo or more hairpin loops (see, e.g., FIGS. 6-8 FIG. 11B ofPCT/US19/14122) or a single hairpin loop (see, e.g., FIG. 10A-10B FIG.11B of PCT/US19/14122). The hairpin loop modified ITR can be generatedby genetic modification of an existing oligo or by de novo biologicaland/or chemical synthesis.

In a non-limiting example, ITR-6 Left and Right (SEQ ID NOS: 111 and112), include 40 nucleotide deletions in the B-B′ and C-C′ arms from thewild-type ITR of AAV2. Nucleotides remaining in the modified ITR arepredicted to form a single hairpin structure. Gibbs free energy ofunfolding the structure is about −54.4 kcal/mol. Other modifications tothe ITR may also be made, including optional deletion of a functionalRep binding site or a TRS site.

Example 3: ceDNA Production Via Oligonucleotide Construction

Another exemplary method of producing a ceDNA vector using a syntheticmethod that involves assembly of various oligonucleotides, is providedin Example 3 of PCT/US19/14122, where a ceDNA vector is produced bysynthesizing a 5′ oligonucleotide and a 3′ ITR oligonucleotide andligating the ITR oligonucleotides to a double-stranded polynucleotidecomprising an expression cassette. FIG. 11B of PCT/US19/14122 shows anexemplary method of ligating a 5′ ITR oligonucleotide and a 3′ ITRoligonucleotide to a double stranded polynucleotide comprising anexpression cassette.

As disclosed herein, the ITR oligonucleotides can comprise WT-ITRs(e.g., see FIG. 3A, FIG. 3C), or modified ITRs (e.g., see, FIG. 3B andFIG. 3D). (See also, e.g., FIGS. 6A, 6B, 7A and 7B of PCT/US19/14122,which is incorporated herein in its entirety). Exemplary ITRoligonucleotides include, but are not limited to SEQ ID NOS: 134-145(e.g., see Table 7 in of PCT/US19/14122). Modified ITRs can includedeletion, insertion, or substitution of one or more nucleotides from thewild-type ITR in the sequences forming B and B′ arm and/or C and C′ arm.ITR oligonucleotides, comprising WT-ITRs or mod-ITRs as describedherein, to be used in the cell-free synthesis, can be generated bygenetic modification or biological and/or chemical synthesis. Asdiscussed herein, the ITR oligonucleotides in Examples 2 and 3 cancomprise WT-ITRs, or modified ITRs (mod-ITRs) in symmetrical orasymmetrical configurations, as discussed herein.

Example 4: ceDNA Production Via a Single-Stranded DNA Molecule

Another exemplary method of producing a ceDNA vector using a syntheticmethod is provided in Example 4 of PCT/US19/14122, and uses asingle-stranded linear DNA comprising two sense ITRs which flank a senseexpression cassette sequence and are attached covalently to twoantisense ITRs which flank an antisense expression cassette, the ends ofwhich single stranded linear DNA are then ligated to form a closed-endedsingle-stranded molecule. One non-limiting example comprisessynthesizing and/or producing a single-stranded DNA molecule, annealingportions of the molecule to form a single linear DNA molecule which hasone or more base-paired regions of secondary structure, and thenligating the free 5′ and 3′ ends to each other to form a closedsingle-stranded molecule.

An exemplary single-stranded DNA molecule for production of a ceDNAvector comprises, from 5′ to 3′: a sense first ITR; a sense expressioncassette sequence; a sense second ITR; an antisense second ITR; anantisense expression cassette sequence; and an antisense first ITR.

A single-stranded DNA molecule for use in the exemplary method ofExample 4 can be formed by any DNA synthesis methodology describedherein, e.g., in vitro DNA synthesis, or provided by cleaving a DNAconstruct (e.g., a plasmid) with nucleases and melting the resultingdsDNA fragments to provide ssDNA fragments.

Annealing can be accomplished by lowering the temperature below thecalculated melting temperatures of the sense and antisense sequencepairs. The melting temperature is dependent upon the specific nucleotidebase content and the characteristics of the solution being used, e.g.,the salt concentration. Melting temperatures for any given sequence andsolution combination are readily calculated by one of ordinary skill inthe art.

The free 5′ and 3′ ends of the annealed molecule can be ligated to eachother, or ligated to a hairpin molecule to form the ceDNA vector.Suitable exemplary ligation methodologies and hairpin molecules aredescribed in Examples 2 and 3.

Example 5: Purifying and/or Confirming Production of ceDNA

Any of the DNA vector products produced by the methods described herein,e.g., including the insect cell based production methods described inExample 1, or synthetic production methods described in Examples 2-4 canbe purified, e.g., to remove impurities, unused components, orbyproducts using methods commonly known by a skilled artisan; and/or canbe analyzed to confirm that DNA vector produced, (in this instance, aceDNA vector) is the desired molecule. An exemplary method forpurification of the DNA vector, e.g., ceDNA is using Qiagen Midi Pluspurification protocol (Qiagen) and/or by gel purification,

The following is an exemplary method for confirming the identity ofceDNA vectors.

ceDNA vectors can be assessed by identified by agarose gelelectrophoresis under native or denaturing conditions as illustrated inFIG. 4D, where (a) the presence of characteristic bands migrating attwice the size on denaturing gels versus native gels after restrictionendonuclease cleavage and gel electrophoretic analysis and (b) thepresence of monomer and dimer (2×) bands on denaturing gels foruncleaved material is characteristic of the presence of ceDNA vector.

Structures of the isolated ceDNA vectors were further analyzed bydigesting the purified DNA with restriction endonucleases selected fora) the presence of only a single cut site within the ceDNA vectors, andb) resulting fragments that were large enough to be seen clearly whenfractionated on a 0.8% denaturing agarose gel (>800 bp). As illustratedin FIGS. 4C and 4D, linear DNA vectors with a non-continuous structureand ceDNA vector with the linear and continuous structure can bedistinguished by sizes of their reaction products—for example, a DNAvector with a non-continuous structure is expected to produce 1 kb and 2kb fragments, while a ceDNA vector with the continuous structure isexpected to produce 2 kb and 4 kb fragments.

Therefore, to demonstrate in a qualitative fashion that isolated ceDNAvectors are covalently closed-ended as is required by definition, thesamples were digested with a restriction endonuclease identified in thecontext of the specific DNA vector sequence as having a singlerestriction site, preferably resulting in two cleavage products ofunequal size (e.g., 1000 bp and 2000 bp). Following digestion andelectrophoresis on a denaturing gel (which separates the twocomplementary DNA strands), a linear, non-covalently closed DNA willresolve at sizes 1000 bp and 2000 bp, while a covalently closed DNA(i.e., a ceDNA vector) will resolve at 2× sizes (2000 bp and 4000 bp),as the two DNA strands are linked and are now unfolded and twice thelength (though single stranded). Furthermore, digestion of monomeric,dimeric, and n-meric forms of the DNA vectors will all resolve as thesame size fragments due to the end-to-end linking of the multimeric DNAvectors (see FIG. 4E).

As used herein, the phrase “assay for the Identification of DNA vectorsby agarose gel electrophoresis under native gel and denaturingconditions” refers to an assay to assess the close-endedness of theceDNA by performing restriction endonuclease digestion followed byelectrophoretic assessment of the digest products. One such exemplaryassay follows, though one of ordinary skill in the art will appreciatethat many art-known variations on this example are possible. Therestriction endonuclease is selected to be a single cut enzyme for theceDNA vector of interest that will generate products of approximately ⅓×and ⅔× of the DNA vector length. This resolves the bands on both nativeand denaturing gels. Before denaturation, it is important to remove thebuffer from the sample. The Qiagen PCR clean-up kit or desalting “spincolumns,” e.g. GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25 columns are someart-known options for the endonuclease digestion. The assay includes forexample, i) digest DNA with appropriate restriction endonuclease(s), 2)apply to e.g., a Qiagen PCR clean-up kit, elute with distilled water,iii) adding 10× denaturing solution (10×=0.5 M NaOH, 10 mM EDTA), add10× dye, not buffered, and analyzing, together with DNA ladders preparedby adding 10× denaturing solution to 4×, on a 0.8-1.0% gel previouslyincubated with 1 mM EDTA and 200 mM NaOH to ensure that the NaOHconcentration is uniform in the gel and gel box, and running the gel inthe presence of 1×denaturing solution (50 mM NaOH, 1 mM EDTA). One ofordinary skill in the art will appreciate what voltage to use to run theelectrophoresis based on size and desired timing of results. Afterelectrophoresis, the gels are drained and neutralized in 1×TBE or TAEand transferred to distilled water or 1×TBE/TAE with 1×SYBR Gold. Bandscan then be visualized with e.g. Thermo Fisher, SYBR® Gold Nucleic AcidGel Stain (10,000× Concentrate in DMSO) and epifluorescent light (blue)or UV (312 nm). The foregoing gel-based method can be adapted topurification purposes by isolating the ceDNA vector from the gel bandand permitting it to renature.

The purity of the generated ceDNA vector can be assessed using anyart-known method. As one exemplary and non-limiting method, contributionof ceDNA-plasmid to the overall UV absorbance of a sample can beestimated by comparing the fluorescent intensity of ceDNA vector to astandard. For example, if based on UV absorbance 4 μg of ceDNA vectorwas loaded on the gel, and the ceDNA vector fluorescent intensity isequivalent to a 2 kb band which is known to be 1 μg, then there is 1 μgof ceDNA vector, and the ceDNA vector is 25% of the total UV absorbingmaterial. Band intensity on the gel is then plotted against thecalculated input that band represents—for example, if the total ceDNAvector is 8 kb, and the excised comparative band is 2 kb, then the bandintensity would be plotted as 25% of the total input, which in this casewould be 0.25 μg for 1.0 μg input. Using the ceDNA vector plasmidtitration to plot a standard curve, a regression line equation is thenused to calculate the quantity of the ceDNA vector band, which can thenbe used to determine the percent of total input represented by the ceDNAvector, or percent purity.

Example 6: Controlled Transgene Expression from ceDNA: TransgeneExpression from the ceDNA Vector In Vivo can be Sustained and/orIncreased by Re-Dose Administration

A ceDNA vector was produced according to the methods described inExample 1 above, using a ceDNA plasmid comprising a CAG promoter (SEQ IDNO: 72) and a luciferase transgene (SEQ ID NO: 56) as an exemplaryFVIII, flanked between asymmetric ITRs (e.g., a 5′ WT-ITR (SEQ ID NO: 2)and a 3′ mod-ITR (SEQ ID NO: 3) and was assessed in different treatmentprograms in vivo. This ceDNA vector was used in all subsequentexperiments described in Examples 6-10. In Example 6, the ceDNA vectorwas purified and formulated with a lipid nanoparticle (LNP ceDNA) andinjected into the tail vein of each CD-1® IGS mice. Liposomes wereformulated with a suitable lipid blend comprising four components toform lipid nanoparticles (LNP) liposomes, including cationic lipids,helper lipids, cholesterol and PEG-lipids.

To assess the sustained expression of the transgene in vivo from theceDNA vector over a long time period, the LNP-ceDNA was administered insterile PBS by tail vein intravenous injection to CD-1® IGS mice ofapproximately 5-7 weeks of age. Three different dosage groups wereassessed: 0.1 mg/kg, 0.5 mg/kg, and 1.0 mg/kg, ten mice per group(except 1.0 mg/kg which had 15 mice per group). Injections wereadministered on day 0. Five mice from each of the groups were injectedwith an additional identical dose on day 28. Luciferase expression wasmeasured by IVIS imaging following intravenous administration into CD-1®IGS mice (Charles River Laboratories; WT mice). Luciferase expressionwas assessed by IVIS imaging following intraperitoneal injection of 150mg/kg luciferin substrate on days 3, 4, 7, 14, 21, 28, 31, 35, and 42,and routinely (e.g., weekly, biweekly or every 10-days or every 2weeks), between days 42-110 days. Luciferase transgene expression as theexemplary FVIII as measured by IVIS imaging for at least 132 days after3 different administration protocols (data not shown).

An extension study was performed to investigate the effect of a re-dose,e.g., a re-administration of LNP-ceDNA expressing luciferase of theLNP-ceDNA treated subjects. In particular, it was assessed to determineif expression levels can be increased by one or more additionaladministrations of the ceDNA vector.

In this study, the biodistribution of luciferase expression from a ceDNAvector was assessed by IVIS in CD-1® IGS mice after an initialintravenous administration of 1.0 mg/kg (i.e., a priming dose) at days 0and 28 (Group A). A second administration of a ceDNA vector wasadministered via tail vein injection of 3 mg/kg (Group B) or 10 mg/kg(Group C) in 1.2 mL in the tail vein at day 84. In this study, five (5)CD-1® mice were used in each of Groups A, B and C. IVIS imaging of themice for luciferase expression was performed prior to the additionaldosing at days 49, 56, 63, and 70 as described above, as well aspost-redose on day 84 and on days 91, 98, 105, 112, and 132. Luciferaseexpression was assessed and detected in all three Groups A, B and Cuntil at least 110 days (the longest time period assessed).

The level of expression of luciferase was shown to be increased by are-dose (i.e., re-administration of the ceDNA composition) of theLNP-ceDNA-Luc, as determined by assessment of luciferase activity in thepresence of luciferin. Luciferase transgene expression as an exemplaryFVIII as measured by IVIS imaging for at least 110 days after 3different administration protocols (Groups A, B and C). The mice thathad not been given any additional redose (1 mg/kg priming dose (i.e.,Group A) treatment had stable luciferase expression observed over theduration of the study. The mice in Group B that had been administered are-dose of 3 mg/kg of the ceDNA vector showed an approximatelyseven-fold increase in observed radiance relative to the mice in GroupC. Surprisingly, the mice re-dosed with 10 mg/kg of the ceDNA vector hada 17-fold increase in observed luciferase radiance over the mice notreceiving any redose (Group A).

Group A shows luciferase expression in CD-1® IGS mice after intravenousadministration of 1 mg/kg of a ceDNA vector into the tail vein at days 0and 28. Group B and C show luciferase expression in CD-1® IGS miceadministered 1 mg/kg of a ceDNA vector at a first time point (day 0) andre-dosed with administration of a ceDNA vector at a second time point of84 days. The second administration (i.e., re-dose) of the ceDNA vectorincreased expression by at least 7-fold, even up to 17-fold.

A 3-fold increase in the dose (i.e., the amount) of ceDNA vector in are-dose administration in Group B (i.e., 3 mg/kg administered atre-dose) resulted in a 7-fold increase in expression of the luciferase.Also, unexpectedly, a 10-fold increase in the amount of ceDNA vector ina re-dose administration (i.e., 10 mg/kg re-dose administered) in GroupC resulted in a 17-fold increase in expression of the luciferase. Thus,the second administration (i.e., re-dose) of the ceDNA increasedexpression by at least 7-fold, even up to 17-fold. This shows that theincrease in transgene expression from the re-dose is greater thanexpected and dependent on the dose or amount of the ceDNA vector in there-dose administration, and appears to be synergistic to the initialtransgene expression from the initial priming administration at day 0.That is, the dose-dependent increase in transgene expression is notadditive, rather, the expression level of the transgene isdose-dependent and greater than the sum of the amount of the ceDNAvector administered at each time point.

Both Groups B and C showed significant dose-dependent increase inexpression of luciferase as compared to control mice (Group A) that werenot re-dosed with a ceDNA vector at the second time point. Takentogether, these data show that the expression of a transgene from ceDNAvector can be increased in a dose-dependent manner by re-dose (i.e.,re-administration) of the ceDNA vector at least a second time point.

Taken together, these data demonstrate that the expression level of atransgene, e.g., FVIII from ceDNA vectors can be maintained at asustained level for at least 84 days and can be increased in vivo aftera redose of the ceDNA vector administered at least at a second timepoint.

Example 7: Sustained Transgene Expression In Vivo of LNP-FormulatedceDNA Vectors

The reproducibility of the results in Example 6 with a different lipidnanoparticle was assessed in vivo in mice. Mice were dosed on day 0 witheither ceDNA vector comprising a luciferase transgene driven by a CAGpromoter that was encapsulated in an LNP different from that used inExample 6 or with that same LNP comprising polyC but lacking ceDNA or aluciferase gene. Specifically, male CD-1® mice of approximately 4 weeksof age were treated with a single injection of 0.5 mg/kgLNP-ceDNA-luciferase or control LNP-polyC, administered intravenouslyvia lateral tail vein on day 0. At day 14 animals were dosedsystemically with luciferin at 150 mg/kg via intraperitoneal injectionat 2.5 mL/kg. At approximately 15 minutes after luciferin administrationeach animal was imaged using an In Vivo Imaging System (“IVIS”).

As shown in FIG. 7, significant fluorescence in the liver was observedin all four ceDNA-treated mice, and very little other fluorescence wasobserved in the animals other than at the injection site, indicatingthat the LNP mediated liver-specific delivery of the ceDNA construct andthat the delivered ceDNA vector was capable of controlled sustainedexpression of its transgene for at least two weeks after administration.

Example 8: Sustained Transgene Expression in the Liver In Vivo fromceDNA Vector Administration

In a separate experiment, the localization of LNP-delivered ceDNA withinthe liver of treated animals was assessed. A ceDNA vector comprising afunctional transgene of interest was encapsulated in the same LNP asused in Example 7 and administered to mice in vivo at a dose level of0.5 mg/kg by intravenous injection. After 6 hours the mice wereterminated and liver samples taken, formalin fixed and paraffin-embeddedusing standard protocols. RNAscope® in situ hybridization assays wereperformed to visualize the ceDNA vectors within the tissue using a probespecific for the ceDNA transgene and detecting using chromogenicreaction and hematoxylin staining (Advanced Cell Diagnostics). FIG. 8shows the results, which indicate that ceDNA is present in hepatocytes.

Example 9: Sustained Ocular Transgene Expression of ceDNA In Vivo

The sustainability of ceDNA vector transgene expression in tissues otherthan the liver was assessed to determine tolerability and expression ofa ceDNA vector after ocular administration in vivo. While luciferase wasused as an exemplary transgene in Example 9, one of ordinary skill canreadily substitute the luciferase transgene with an FVIII sequence fromany of those listed in Table 1.

On day 0, male Sprague Dawley rats of approximately 9 weeks of age wereinjected sub-retinally with 5 μL of either ceDNA vector comprising aluciferase transgene formulated with jetPEI® transfection reagent(Polyplus) or plasmid DNA encoding luciferase formulated with jetPEI®,both at a concentration of 0.25 μg/μL. Four rats were tested in eachgroup Animals were sedated and injected sub-retinally in the right eyewith the test article using a 33-gauge needle. The left eye of eachanimal was untreated. Immediately after injection eyes were checked withoptical coherence tomography or fundus imaging in order to confirm thepresence of a subretinal bleb. Rats were treated with buprenorphine andtopical antibiotic ointment according to standard procedures.

At days 7, 14, 21, 28, and 35, the animals in both groups were dosedsystemically with freshly made luciferin at 150 mg/kg viaintraperitoneal injection at 2.5 mL/kg. at 5-15 minutes post luciferinadministration, all animals were imaged using IVIS while underisoflurane anesthesia. Total Flux [p/s] and average Flux (p/s/sr/cm²) ina region of interest encompassing the eye were obtained over 5 minutesof exposure. The results were graphed as average radiance of eachtreatment group in the treated eye (“injected”) relative to the averageradiance of each treatment group in the untreated eye (“uninjected”)(FIG. 9B). Significant fluorescence was readily detectable in the ceDNAvector-treated eyes but much weaker in the plasmid-treated eyes (FIG.9A). After 35 days, the plasmid-injected rats were terminated, while thestudy continued for the ceDNA-treated rats, with luciferin injection andIVIS imaging at days 42, 49, 56, 63, 70, and 99. The results demonstratethat ceDNA vector introduced in a single injection to rat eye mediatedtransgene expression in vivo and that that expression was sustained at ahigh level at least through 99 days after injection.

Example 10: Sustained Dosing and Redosing of ceDNA Vector in Rag2 Mice

In situations where one or more of the transgenes encoded in the geneexpression cassette of the ceDNA vector is expressed in a hostenvironment (e.g., cell or subject) where the expressed protein isrecognized as foreign, the possibility exists that the host will mountan adaptive immune response that may result in undesired depletion ofthe expression product, which could potentially be confused for lack ofexpression. In some cases, this may occur with a reporter molecule thatis heterologous to the normal host environment. Accordingly, ceDNAvector transgene expression was assessed in vivo in the Rag2 mouse modelwhich lacks B and T cells and therefore does not mount an adaptiveimmune response to non-native murine proteins such as luciferase.Briefly, c57bl/6 and Rag2 knockout mice were dosed intravenously viatail vein injection with 0.5 mg/kg of LNP-encapsulated ceDNA vectorexpressing luciferase or a polyC control at day 0, and at day 21 certainmice were redosed with the same LNP-encapsulated ceDNA vector at thesame dose level. All testing groups consisted of 4 mice each. IVISimaging was performed after luciferin injection as described in Example9 at weekly intervals.

Comparing the total flux observed from the IVIS analyses, thefluorescence observed in the wild-type mice (an indirect measure of thepresence of expressed luciferase) dosed with LNP-ceDNA vector-Lucdecreased gradually after day 21 whereas the Rag2 mice administered thesame treatment displayed relatively constant sustained expression ofluciferase over the 42 day experiment (FIG. 10A). The approximately21-day time point of the observed decrease in the wild-type micecorresponds to the timeframe in which an adapative immune response mightexpect to be produced. Re-administration of the LNP-ceDNA vector in theRag2 mice resulted in a marked increase in expression which wassustained over the at least 21 days it was tracked in this study (FIG.10B). The results suggest that adaptive immunity may play a role when anon-native protein is expressed from a ceDNA vector in a host, and thatobserved decreases in expression in the 20+ day timeframe from initialadministration may signal a confounding adaptive immune response to theexpressed molecule rather than (or in addition to) a decline inexpression. Of note, this response is expected to be low when expressingnative proteins in a host where it is anticipated that the host willproperly recognize the expressed molecules as self and will not developsuch an immune response.

Example 11: Impact of Liver-Specific Expression and CpG Modulation onSustained Expression

As described in Example 10, undesired host immune response may in somecases artificially dampen what would otherwise be sustained expressionof one or more desired transgenes from an introduced ceDNA vector. Twoapproaches were taken to assess the impact of avoiding and/or dampeningpotential host immune response on sustained expression from a ceDNAvector. First, since the ceDNA-Luc vector used in the preceding exampleswas under the control of a constitutive CAG promoter, a similarconstruct was made using a liver-specific promoter (hAAT) or a differentconstitutive promoter (hEF-1) to see whether avoiding prolonged exposureto myeloid cells or non-liver tissue reduced any observed immuneeffects. Second, certain of the ceDNA-luciferase constructs wereengineered to be reduced in CpG content, a known trigger for host immunereaction. ceDNA-encoded luciferase gene expression upon administrationof such engineered and promoter-switched ceDNA vectors to mice wasmeasured.

Three different ceDNA vectors were used, each encoding luciferase as thetransgene. The first ceDNA vector had a high number of unmethylated CpG(˜350) and comprised the constitutive CAG promoter (“ceDNA CAG”); thesecond had a moderate number of unmethylated CpG (˜60) and comprised theliver-specific hAAT promoter (“ceDNA hAAT low CpG”); and the third was amethylated form of the second, such that it contained no unmethylatedCpG and also comprised the hAAT promoter (“ceDNA hAAT No CpG”). TheceDNA vectors were otherwise identical. The vectors were prepared asdescribed above.

Four groups of four male CD-1® mice, approximately 4 weeks old, weretreated with one of the ceDNA vectors encapsulated in an LNP or a polyCcontrol. On day 0 each mouse was administered a single intravenous tailvein injection of 0.5 mg/kg ceDNA vector in a volume of 5 mL/kg. Bodyweights were recorded on days −1, −, 1, 2, 3, 7, and weekly thereafteruntil the mice were terminated. Whole blood and serum samples were takenon days 0, 1, and 35. In-life imaging was performed on days 7, 14, 21,28, and 35, and weekly thereafter using an in vivo imaging system(IVIS). For the imaging, each mouse was injected with luciferin at 150mg/kg via intraperitoneal injection at 2.5 mL/kg. After 15 minutes, eachmouse was anaesthetized and imaged. The mice were terminated at day 93and terminal tissues collected, including liver and spleen. Cytokinemeasurements were taken 6 hours after dosing on day 0.

While all of the ceDNA-treated mice displayed significant fluorescenceat days 7 and 14, the fluorescence decreased rapidly in the ceDNA CAGmice after day 14 and more gradually decreased for the remainder of thestudy. In contrast, the total flux for the ceDNA hAAT low CpG and NoCpG-treated mice remained at a steady high level (FIG. 11). Thissuggested that directing the ceDNA vector delivery specifically to theliver resulted in sustained, durable transgene expression from thevector over at least 77 days after a single injection. Constructs thatwere CpG minimized or completely absent of CpG content had similardurable sustained expression profiles, while the high CpG constitutivepromoter construct exhibited a decline in expression over time,suggesting that host immune activation by the ceDNA vector introductionmay play a role in any decreased expression observed from such vector ina subject. These results provide alternative methods of tailoring theduration of the response to the desired level by selecting atissue-restricted promoter and/or altering the CpG content of the ceDNAvector in the event that a host immune response is observed—apotentially transgene-specific response.

Example 12: Hydrodynamic Delivery of ceDNA Expressing FVIII

A well-known method of introducing nucleic acid to the liver in rodentsis by hydrodynamic tail vein injection. In this system, the pressurizedinjection in a large volume of non-encapsulated nucleic acid results ina transient increase in cell permeability and delivery directly intotissues and cells. This provides an experimental mechanism to bypassmany of the host immune systems, such as macrophage delivery, providingthe opportunity to observe delivery and expression in the absence ofsuch activity.

Three different ceDNA vectors, each with a wild-type left ITR and atruncation mutant right ITR and having a transgene region encodingFVIII, were prepared and purified as described above in Examples 1 and5. ceDNA FVIII vectors under the control of a liver-specific promoter orPBS alone were administered to male C57bl/6J mice of approximately 6weeks of age. The naked ceDNA vectors were dosed at 0.005 mg per animal(4 animals per group) by hydrodynamic intravenous injection via lateraltail vein in a volume of either 1.2 mL administered over 5 seconds or ata dose of 100 mL/kg administered over 5-8 seconds. Body weights weremeasured on days 0, 1, 2, 3 and 7 and weekly thereafter. Blood sampleswere collected from each treated animal on days 3, 7, 14, 21, and atterminal day 28. The presence of expressed FVIII in the plasma sampleswas measured by the Human Total Factor VIII ELISA kit (InnovativeResearch).

As shown in FIG. 11A, FVIII was readily detected in day 3 and 7 plasmasamples from mice treated with two of the three ceDNA FVIII vectors, butwas not observed in mice treated with PBS. FIG. 11B shows that FVIIIexpression was dose-volume dependent in the two vectors where FVIIIexpression was observed, and remained relatively constant over the28-day study with the exception of vector 3, which decreased rapidly inexpression after day 7. This experiment demonstrated that ceDNA vectorswere able to express FVIII from the liver after hydrodynamic injection,and that FVIII was rapidly and readily detectable in the serum afterceDNA administration.

Example 13: Hydrodynamic Delivery of ceDNA Expressing FVIII-Testing ofVarious ceDNA Vectors Expressing FVIII

A hydrodynamic delivery system as described in Example 12 was used totest the effect of various ceDNA vectors expressing FVIII on serum FVIIIlevels, where detection of FVIII in the serum indicated that the ceDNAvector was able to express FVIII after injection.

ceDNA vectors as described in Example 12 were prepared. Each of theceDNA FVIII vectors (alone, without any LNP encapsulation) and thecontrol were administered by hydrodynamic intravenous (IV) route ofinjection to C57BL/6 mice. The ceDNA vectors were dosed at 5 μg peranimal (4 animals per group) by hydrodynamic intravenous injection vialateral tail vein in a volume of 90-100 mg/mL. The terminal timepointwas day 7. Vehicle alone was used as control.

The following ceDNA vectors were tested in three separate experiments.Sequences for the below ceDNA vectors are shown in Table 9. In Table 9,the open reading frame is shown by underlining The promoter is shown inbold.

(1) ceDNAFVIII-vector 4, ceDNAFVIII-vector 6, ceDNAFVIII-vector 12,ceDNAFVIII-vector 14, ceDNAFVIII-vector 16, ceDNAFVIII-vector 18,ceDNAFVIII-vector 19, ceDNAFVIII-vector 21, ceDNAFVIII-vector 22;(2) ceDNAFVIII-vector 1, ceDNAFVIII-vector 2, ceDNAFVIII-vector 3,ceDNAFVIII-vector 5, ceDNAFVIII-vector 7, ceDNAFVIII-vector 8,ceDNAFVIII-vector 12, ceDNAFVIII-vector 13, ceDNAFVIII-vector 15;(3) ceDNAFVIII-vector 9, ceDNAFVIII-vector 10, ceDNAFVIII-vector 11,ceDNAFVIII-vector 12, ceDNAFVIII-vector 17, ceDNAFVIII-vector 20,ceDNAFVIII-vector 24, ceDNAFVIII-vector 25, ceDNAFVIII-vector 26.

TABLE 9 ceDNA Vector Name Nucleic Acid Sequence ceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 1TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT192)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGGAGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAAAGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATTTTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGTGCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTAGAGGTACCGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCTGGAATCCGGCCTGATGAACAGCCAAGAGTCCTCCTGGGGCAAGAATGTGTCCAGCACCGAGTCCGGCAGACTGTTCAAGGGAAAGAGAGCCCACGGACCTGCTCTGCTGACCAAGGATAACGCCCTGTTCAAAGTGTCCATCAGCCTGCTCAAGACCAACAAGACCTCCAACAACTCCGCCACCAACAGAAAGACCCACATCGACGGCCCTAGCCTGCTGATCGAGAATAGCCCTAGCGTCTGGCAGAATATCCTGGAAAGCGACACCGAGTTCAAGAAAGTGACCCCTCTGATCCACGACCGGATGCTCATGGACAAGAACGCCACCGCTCTGCGGCTGAACCACATGAGCAACAAGACAACCAGCAGCAAGAATATGGAAATGGTGCAGCAGAAGAAAGAGGGCCCCATTCCTCCAGACGCTCAGAACCCCGATATGAGCTTCTTCAAGATGCTCTTTCTGCCCGAGAGCGCCCGGTGGATTCAGAGAACACACGGCAAGAACTCCCTGAACTCCGGCCAGGGACCTTCTCCAAAGCAGCTGGTTTCCCTGGGACCTGAGAAGTCCGTGGAAGGACAGAACTTCCTGAGCGAAAAGAACAAAGTGGTCGTCGGCAAGGGCGAGTTCACCAAGGATGTGGGCCTGAAAGAGATGGTCTTTCCCAGCAGCCGGAACCTGTTCCTGACCAACCTGGACAACCTGCACGAGAACAACACCCACAATCAAGAGAAGAAGATCCAAGAGGAAATCGAAAAGAAAGAGACACTCATCCAAGAGAACGTGGTGCTGCCTCAGATCCACACAGTGACCGGCACCAAGAACTTTATGAAGAATCTGTTCCTGCTGAGTACCCGGCAGAACGTGGAAGGCAGCTACGATGGCGCTTATGCCCCTGTGCTGCAGGACTTCAGATCCCTGAACGACTCCACCAATCGGACAAAGAAGCACACAGCCCACTTCTCCAAGAAGGGCGAAGAAGAGAACCTGGAAGGACTGGGCAATCAGACCAAGCAGATCGTCGAGAAGTACGCCTGCACCACCAGAATCAGCCCCAACACAAGCCAGCAGAACTTCGTGACCCAGCGGAGCAAAAGAGCCCTGAAGCAGTTTCGGCTGCCCCTGGAAGAAACCGAGCTGGAAAAGCGGATCATCGTGGACGACACCAGCACACAGTGGTCCAAGAACATGAAGCACTTGACCCCTAGCACACTGACCCAGATCGACTACAACGAGAAAGAGAAGGGCGCTATCACACAGAGCCCACTGAGCGACTGTCTGACCAGAAGCCACAGCATCCCTCAGGCCAACAGATCCCCTCTGCCAATCGCCAAAGTGTCTAGCTTCCCCAGCATCAGACCCATCTACCTGACCAGAGTGCTGTTCCAGGACAACAGCAGCCATCTGCCAGCCGCCAGCTACCGGAAGAAAGATTCTGGCGTGCAAGAGAGCAGCCACTTTCTGCAGGGCGCTAAGAAGAACAATCTGAGCCTGGCTATTCTGACCCTGGAAATGACCGGCGATCAGAGAGAAGTCGGCTCTCTGGGCACCAGCGCCACAAATAGCGTGACCTACAAAAAGGTGGAAAACACCGTGCTGCCTAAGCCTGACCTGCCAAAGACAAGCGGCAAGGTGGAACTGCTGCCAAAGGTGCACATCTACCAGAAGGACCTGTTTCCTACCGAGACAAGCAACGGCTCTCCCGGCCATCTGGATCTGGTGGAAGGATCTCTGCTGCAGGGAACCGAGGGCGCCATCAAGTGGAACGAGGCCAATAGACCTGGCAAGGTGCCCTTCCTGAGAGTGGCCACAGAGTCCAGCGCCAAGACACCCTCTAAACTGCTGGACCCTCTGGCCTGGGACAACCACTATGGCACTCAGATCCCCAAAGAGGAATGGAAGTCCCAAGAGAAGTCCCCTGAAAAGACCGCCTTCAAGAAGAAGGACACCATTCTGTCCCTGAATGCCTGCGAGAGCAACCACGCCATTGCCGCCATCAATGAGGGCCAGAACAAGCCCGAGATCGAAGTGACCTGGGCCAAGCAGGGAAGAACCGAGAGACTGTGTAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGAGAGAAATCACCCGGACCACACTGCAGAGCGACCAAGAAGAGATCGATTACGACGATACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCTCGGAGCTTCCAGAAGAAAACCAGGCACTACTTTATTGCCGCCGTCGAGCGGCTGTGGGACTACGGAATGTCTAGCTCTCCTCACGTGCTGCGGAATAGAGCCCAGTCTGGTAGCGTGCCCCAGTTCAAAAAGGTCGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCAGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCATATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGACCTCGAAAAGGACGTGCACAGCGGACTCATCGGCCCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTTTTCACCATCTTCGACGAAACGAAGTCCTGGTACTTCACCGAAAACATGGAAAGAAACTGCAGGGCCCCTTGCAACATTCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACTCCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTTGAGATGCTGCCTAGCAAGGCCGGAATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGGATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGCCACATTAGAGACTTCCAGATCACCGCCAGCGGACAGTATGGACAGTGGGCCCCTAAACTGGCCAGACTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCTTTCAGCTGGATCAAAGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAAAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTGATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCAGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCTCTAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCTAAGGCCCGGCTTCATCTGCAAGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAATGGCTCCAGGTGGACTTTCAGAAAACCATGAAAGTGACAGGCGTGACCACACAGGGCGTCAAGTCCCTGCTGACCTCTATGTACGTGAAAGAGTTTCTGATCAGCTCCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGAAATCAGGACAGCTTCACACCCGTGGTCAATAGTCTGGACCCACCACTGCTGACCCGCTACCTGCGAATTCACCCTCAGTCTTGGGTGCACCAGATTGCCCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGG ceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 2TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT193)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGGAGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAAAGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATTTTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGTGCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTAGAGGTACCGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGG ceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 3TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT194)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCTGGAATCCGGCCTGATGAACAGCCAAGAGTCCTCCTGGGGCAAGAATGTGTCCAGCACCGAGTCCGGCAGACTGTTCAAGGGAAAGAGAGCCCACGGACCTGCTCTGCTGACCAAGGATAACGCCCTGTTCAAAGTGTCCATCAGCCTGCTCAAGACCAACAAGACCTCCAACAACTCCGCCACCAACAGAAAGACCCACATCGACGGCCCTAGCCTGCTGATCGAGAATAGCCCTAGCGTCTGGCAGAATATCCTGGAAAGCGACACCGAGTTCAAGAAAGTGACCCCTCTGATCCACGACCGGATGCTCATGGACAAGAACGCCACCGCTCTGCGGCTGAACCACATGAGCAACAAGACAACCAGCAGCAAGAATATGGAAATGGTGCAGCAGAAGAAAGAGGGCCCCATTCCTCCAGACGCTCAGAACCCCGATATGAGCTTCTTCAAGATGCTCTTTCTGCCCGAGAGCGCCCGGTGGATTCAGAGAACACACGGCAAGAACTCCCTGAACTCCGGCCAGGGACCTTCTCCAAAGCAGCTGGTTTCCCTGGGACCTGAGAAGTCCGTGGAAGGACAGAACTTCCTGAGCGAAAAGAACAAAGTGGTCGTCGGCAAGGGCGAGTTCACCAAGGATGTGGGCCTGAAAGAGATGGTCTTTCCCAGCAGCCGGAACCTGTTCCTGACCAACCTGGACAACCTGCACGAGAACAACACCCACAATCAAGAGAAGAAGATCCAAGAGGAAATCGAAAAGAAAGAGACACTCATCCAAGAGAACGTGGTGCTGCCTCAGATCCACACAGTGACCGGCACCAAGAACTTTATGAAGAATCTGTTCCTGCTGAGTACCCGGCAGAACGTGGAAGGCAGCTACGATGGCGCTTATGCCCCTGTGCTGCAGGACTTCAGATCCCTGAACGACTCCACCAATCGGACAAAGAAGCACACAGCCCACTTCTCCAAGAAGGGCGAAGAAGAGAACCTGGAAGGACTGGGCAATCAGACCAAGCAGATCGTCGAGAAGTACGCCTGCACCACCAGAATCAGCCCCAACACAAGCCAGCAGAACTTCGTGACCCAGCGGAGCAAAAGAGCCCTGAAGCAGTTTCGGCTGCCCCTGGAAGAAACCGAGCTGGAAAAGCGGATCATCGTGGACGACACCAGCACACAGTGGTCCAAGAACATGAAGCACTTGACCCCTAGCACACTGACCCAGATCGACTACAACGAGAAAGAGAAGGGCGCTATCACACAGAGCCCACTGAGCGACTGTCTGACCAGAAGCCACAGCATCCCTCAGGCCAACAGATCCCCTCTGCCAATCGCCAAAGTGTCTAGCTTCCCCAGCATCAGACCCATCTACCTGACCAGAGTGCTGTTCCAGGACAACAGCAGCCATCTGCCAGCCGCCAGCTACCGGAAGAAAGATTCTGGCGTGCAAGAGAGCAGCCACTTTCTGCAGGGCGCTAAGAAGAACAATCTGAGCCTGGCTATTCTGACCCTGGAAATGACCGGCGATCAGAGAGAAGTCGGCTCTCTGGGCACCAGCGCCACAAATAGCGTGACCTACAAAAAGGTGGAAAACACCGTGCTGCCTAAGCCTGACCTGCCAAAGACAAGCGGCAAGGTGGAACTGCTGCCAAAGGTGCACATCTACCAGAAGGACCTGTTTCCTACCGAGACAAGCAACGGCTCTCCCGGCCATCTGGATCTGGTGGAAGGATCTCTGCTGCAGGGAACCGAGGGCGCCATCAAGTGGAACGAGGCCAATAGACCTGGCAAGGTGCCCTTCCTGAGAGTGGCCACAGAGTCCAGCGCCAAGACACCCTCTAAACTGCTGGACCCTCTGGCCTGGGACAACCACTATGGCACTCAGATCCCCAAAGAGGAATGGAAGTCCCAAGAGAAGTCCCCTGAAAAGACCGCCTTCAAGAAGAAGGACACCATTCTGTCCCTGAATGCCTGCGAGAGCAACCACGCCATTGCCGCCATCAATGAGGGCCAGAACAAGCCCGAGATCGAAGTGACCTGGGCCAAGCAGGGAAGAACCGAGAGACTGTGTAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGAGAGAAATCACCCGGACCACACTGCAGAGCGACCAAGAAGAGATCGATTACGACGATACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCTCGGAGCTTCCAGAAGAAAACCAGGCACTACTTTATTGCCGCCGTCGAGCGGCTGTGGGACTACGGAATGTCTAGCTCTCCTCACGTGCTGCGGAATAGAGCCCAGTCTGGTAGCGTGCCCCAGTTCAAAAAGGTCGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCAGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCATATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGACCTCGAAAAGGACGTGCACAGCGGACTCATCGGCCCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTTTTCACCATCTTCGACGAAACGAAGTCCTGGTACTTCACCGAAAACATGGAAAGAAACTGCAGGGCCCCTTGCAACATTCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACTCCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTTGAGATGCTGCCTAGCAAGGCCGGAATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGGATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGCCACATTAGAGACTTCCAGATCACCGCCAGCGGACAGTATGGACAGTGGGCCCCTAAACTGGCCAGACTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCTTTCAGCTGGATCAAAGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAAAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTGATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCAGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCTCTAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCTAAGGCCCGGCTTCATCTGCAAGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAATGGCTCCAGGTGGACTTTCAGAAAACCATGAAAGTGACAGGCGTGACCACACAGGGCGTCAAGTCCCTGCTGACCTCTATGTACGTGAAAGAGTTTCTGATCAGCTCCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGAAATCAGGACAGCTTCACACCCGTGGTCAATAGTCTGGACCCACCACTGCTGACCCGCTACCTGCGAATTCACCCTCAGTCTTGGGTGCACCAGATTGCCCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGG ceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 4TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT195)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 5TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT196)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCTGGAATCCGGCCTGATGAACAGCCAAGAGTCCTCCTGGGGCAAGAATGTGTCCAGCACCGAGTCCGGCAGACTGTTCAAGGGAAAGAGAGCCCACGGACCTGCTCTGCTGACCAAGGATAACGCCCTGTTCAAAGTGTCCATCAGCCTGCTCAAGACCAACAAGACCTCCAACAACTCCGCCACCAACAGAAAGACCCACATCGACGGCCCTAGCCTGCTGATCGAGAATAGCCCTAGCGTCTGGCAGAATATCCTGGAAAGCGACACCGAGTTCAAGAAAGTGACCCCTCTGATCCACGACCGGATGCTCATGGACAAGAACGCCACCGCTCTGCGGCTGAACCACATGAGCAACAAGACAACCAGCAGCAAGAATATGGAAATGGTGCAGCAGAAGAAAGAGGGCCCCATTCCTCCAGACGCTCAGAACCCCGATATGAGCTTCTTCAAGATGCTCTTTCTGCCCGAGAGCGCCCGGTGGATTCAGAGAACACACGGCAAGAACTCCCTGAACTCCGGCCAGGGACCTTCTCCAAAGCAGCTGGTTTCCCTGGGACCTGAGAAGTCCGTGGAAGGACAGAACTTCCTGAGCGAAAAGAACAAAGTGGTCGTCGGCAAGGGCGAGTTCACCAAGGATGTGGGCCTGAAAGAGATGGTCTTTCCCAGCAGCCGGAACCTGTTCCTGACCAACCTGGACAACCTGCACGAGAACAACACCCACAATCAAGAGAAGAAGATCCAAGAGGAAATCGAAAAGAAAGAGACACTCATCCAAGAGAACGTGGTGCTGCCTCAGATCCACACAGTGACCGGCACCAAGAACTTTATGAAGAATCTGTTCCTGCTGAGTACCCGGCAGAACGTGGAAGGCAGCTACGATGGCGCTTATGCCCCTGTGCTGCAGGACTTCAGATCCCTGAACGACTCCACCAATCGGACAAAGAAGCACACAGCCCACTTCTCCAAGAAGGGCGAAGAAGAGAACCTGGAAGGACTGGGCAATCAGACCAAGCAGATCGTCGAGAAGTACGCCTGCACCACCAGAATCAGCCCCAACACAAGCCAGCAGAACTTCGTGACCCAGCGGAGCAAAAGAGCCCTGAAGCAGTTTCGGCTGCCCCTGGAAGAAACCGAGCTGGAAAAGCGGATCATCGTGGACGACACCAGCACACAGTGGTCCAAGAACATGAAGCACTTGACCCCTAGCACACTGACCCAGATCGACTACAACGAGAAAGAGAAGGGCGCTATCACACAGAGCCCACTGAGCGACTGTCTGACCAGAAGCCACAGCATCCCTCAGGCCAACAGATCCCCTCTGCCAATCGCCAAAGTGTCTAGCTTCCCCAGCATCAGACCCATCTACCTGACCAGAGTGCTGTTCCAGGACAACAGCAGCCATCTGCCAGCCGCCAGCTACCGGAAGAAAGATTCTGGCGTGCAAGAGAGCAGCCACTTTCTGCAGGGCGCTAAGAAGAACAATCTGAGCCTGGCTATTCTGACCCTGGAAATGACCGGCGATCAGAGAGAAGTCGGCTCTCTGGGCACCAGCGCCACAAATAGCGTGACCTACAAAAAGGTGGAAAACACCGTGCTGCCTAAGCCTGACCTGCCAAAGACAAGCGGCAAGGTGGAACTGCTGCCAAAGGTGCACATCTACCAGAAGGACCTGTTTCCTACCGAGACAAGCAACGGCTCTCCCGGCCATCTGGATCTGGTGGAAGGATCTCTGCTGCAGGGAACCGAGGGCGCCATCAAGTGGAACGAGGCCAATAGACCTGGCAAGGTGCCCTTCCTGAGAGTGGCCACAGAGTCCAGCGCCAAGACACCCTCTAAACTGCTGGACCCTCTGGCCTGGGACAACCACTATGGCACTCAGATCCCCAAAGAGGAATGGAAGTCCCAAGAGAAGTCCCCTGAAAAGACCGCCTTCAAGAAGAAGGACACCATTCTGTCCCTGAATGCCTGCGAGAGCAACCACGCCATTGCCGCCATCAATGAGGGCCAGAACAAGCCCGAGATCGAAGTGACCTGGGCCAAGCAGGGAAGAACCGAGAGACTGTGTAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGAGAGAAATCACCCGGACCACACTGCAGAGCGACCAAGAAGAGATCGATTACGACGATACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCTCGGAGCTTCCAGAAGAAAACCAGGCACTACTTTATTGCCGCCGTCGAGCGGCTGTGGGACTACGGAATGTCTAGCTCTCCTCACGTGCTGCGGAATAGAGCCCAGTCTGGTAGCGTGCCCCAGTTCAAAAAGGTCGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCAGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCATATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGACCTCGAAAAGGACGTGCACAGCGGACTCATCGGCCCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTTTTCACCATCTTCGACGAAACGAAGTCCTGGTACTTCACCGAAAACATGGAAAGAAACTGCAGGGCCCCTTGCAACATTCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACTCCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTTGAGATGCTGCCTAGCAAGGCCGGAATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGGATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGCCACATTAGAGACTTCCAGATCACCGCCAGCGGACAGTATGGACAGTGGGCCCCTAAACTGGCCAGACTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCTTTCAGCTGGATCAAAGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAAAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTGATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCAGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCTCTAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCTAAGGCCCGGCTTCATCTGCAAGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAATGGCTCCAGGTGGACTTTCAGAAAACCATGAAAGTGACAGGCGTGACCACACAGGGCGTCAAGTCCCTGCTGACCTCTATGTACGTGAAAGAGTTTCTGATCAGCTCCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGAAATCAGGACAGCTTCACACCCGTGGTCAATAGTCTGGACCCACCACTGCTGACCCGCTACCTGCGAATTCACCCTCAGTCTTGGGTGCACCAGATTGCCCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGG ceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 6TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT197)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGG ceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 7TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT198)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCCTCCTGTGCTGAAGCGGCACCAGCGGGAAATCACCAGAACCACACTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGATCCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGGGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCAAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATTAGCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGceDNAFVIII-GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCvector 8TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC(SEQ ID NO:TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT199)AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGCGGCCGCGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGAAGCTTCAGCCAGAATAGCAGACACCCCTCCACCAGACAGAAGCAGTTCAACGCCACAACAATCCCCGAGAACGACATCGAGAAAACCGATCCTTGGTTTGCCCACAGAACCCCTATGCCTAAGATCCAGAACGTGTCCTCCAGCGATCTGCTGATGCTCCTGAGACAGAGCCCTACACCTCACGGACTGAGCCTGTCCGATCTGCAAGAGGCCAAATACGAAACCTTCAGCGACGACCCTTCTCCTGGCGCCATCGACAGCAACAATAGCCTGAGCGAGATGACCCACTTCAGACCACAGCTGCACCACAGCGGCGACATGGTGTTTACACCTGAGAGCGGCCTCCAGCTGAGACTGAATGAGAAGCTGGGAACCACCGCCGCCACCGAGCTGAAGAAACTGGACTTCAAGGTGTCCTCTACCAGCAACAACCTGATCAGCACAATCCCCTCCGACAACCTGGCTGCCGGCACCGACAACACATCTTCTCTGGGCCCACCTAGCATGCCCGTGCACTACGATAGCCAGCTGGATACCACACTGTTCGGCAAGAAGTCTAGCCCTCTGACAGAGTCTGGCGGCCCTCTGTCTCTGAGCGAGGAAAACAACGACAGCAAGCTGCCTCCTGTGCTGAAGCGGCACCAGCGGGAAATCACCAGAACCACACTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGATCCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCTCCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCCGGAAGAATTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGGGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACTCCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACTCCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCAAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGAAAGAAGTGGCAGACCTACCGGGGCAATAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATTAGCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGG ceDNAFVIII-AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACATAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATvector 9AGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATA(SEQ ID NO:CGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCCTAGGCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTG200)AGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCGCGGCCGCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTTTAGCCTTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTGTCGACAGAATTCCTCGAAGATCCGAAGGGGTTCAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCATCGATTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACAGTTAACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCGGCGCGCCGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGTCGACGCTAGCAGCTGATGCATAGCATGCGGTACCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACTACGGGTACGTAGGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACGGCCGAGGTCTACCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGCAGGGCATCCATTTATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCTGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCAAATAAGCGTTGATATTCAGTCAATTACAAACATTAATAACGAAGAGATGACAGAAAAATTTTCATTCTGTGACAGAGAA ceDNAFVIII-AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACATAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATvector 10AGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATA(SEQ ID NO:CGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCCTAGGCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTG201)AGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCGCGGCCGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGGAGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAAAGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATTTTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGTGCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTAGAGGTACCGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCATCGATTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACAGTTAACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCGGCGCGCCGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGTCGACGCTAGCAGCTGATGCATAGCATGCGGTACCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACTACGGGTACGTAGGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACGGCCGAGGTCTACCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGCAGGGCATCCATTTATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCTGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCAAATAAGCGTTGATATTCAGTCAATTACAAACATTAATAACGAAGAGATGACAGAAAAATTTTCATTCTGTGACAGAGAA ceDNAFVIII-AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACATAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATvector 11AGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATA(SEQ ID NO:CGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCCTAGGCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTG202)AGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCGCGGCCGCCGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGTTTAAACCGCAGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGCCATGGTGCTAGCAGCTGATGCATAGCATGCGGTACCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACTACGGGTACGTAGGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACGGCCGAGGTCTACCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGCAGGGCATCCATTTATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCTGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCAAATAAGCGTTGATATTCAGTCAATTACAAACATTAATAACGAAGAGATGACAGAAAAATTTTCATTCTGTGACAGAGAAceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 12GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAGTTTGCTGCTTGCAATGTTTGCCCATTTTAGGGTGGACACAGGACGCTGTGGTTTCTGAGCCAGGGGGCGACTCAGATCCCAGCCAGTGGAC203)TTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAACCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGAAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 13GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTGACTTAGGTTACTTATTCTCCTTTTG204)TTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGCTAGCAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTGACACTGACATCCACTTTTTCTTTTTCTCCACAGGTTTAAACGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGTCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGGTATTACCTGGGGGCTGTGGAGCTGAGCTGGGACTATATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCTAGGTTCCCCCCCAGGGTGCCCAAGAGCTTCCCCTTTAACACTTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGGCTGCTGGGGCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCTTCTGAGGGGGCTGAGTATGATGACCAGACTAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTTCCTGGGGGCAGCCATACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGTCTCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCTCTGCTGGTGTGTAGGGAGGGCAGCCTGGCTAAGGAAAAGACCCAGACCCTGCATAAGTTTATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAGACCAAGAACAGCCTGATGCAGGATAGGGATGCTGCCTCTGCCAGGGCTTGGCCTAAGATGCACACTGTGAATGGGTATGTGAATAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGGATGGGCACCACCCCTGAGGTCCATAGCATCTTCCTGGAGGGCCACACTTTCCTGGTGAGGAACCACAGACAGGCCTCTCTGGAGATCTCTCCCATCACCTTCCTGACTGCTCAGACTCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTTTGCCATATTAGCAGCCACCAGCATGATGGGATGGAGGCCTATGTGAAGGTGGATAGCTGCCCTGAGGAGCCTCAGCTGAGGATGAAGAACAATGAGGAGGCTGAAGACTATGATGATGACCTGACTGATTCTGAGATGGATGTGGTGAGGTTTGATGATGACAATAGCCCCAGCTTCATTCAGATCAGGTCTGTGGCCAAGAAACACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAAGAGGACTGGGACTATGCTCCCCTGGTGCTGGCCCCTGATGATAGGTCTTATAAGAGCCAGTACCTGAACAATGGGCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTGAGGTTCATGGCCTACACTGATGAAACCTTCAAAACCAGGGAGGCCATTCAGCATGAGTCTGGCATCCTGGGCCCTCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTATCCTCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAAGACTTCCCCATCCTGCCTGGGGAGATCTTTAAGTATAAGTGGACTGTGACTGTGGAGGATGGCCCTACCAAGTCTGACCCCAGGTGTCTGACCAGGTACTATTCTAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGGCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTTTCTGTGTTTGATGAGAATAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTTCTGCCCAATCCTGCTGGGGTGCAGCTGGAGGATCCTGAGTTCCAGGCCAGCAATATCATGCATAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTTCTGTCTGTGTTCTTTTCTGGCTATACCTTCAAGCACAAGATGGTGTATGAGGATACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAATCCTGGGCTGTGGATCCTGGGGTGCCACAACTCTGATTTTAGGAACAGGGGGATGACTGCCCTGCTGAAGGTGTCTAGCTGTGATAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATTTCTGCTTATCTGCTGTCTAAGAATAATGCCATTGAGCCCAGAAGCTTCAGCCAGAATCCCCCTGTGCTGAAGAGACATCAGAGGGAGATCACCAGAACTACCCTGCAGTCTGATCAGGAGGAGATTGACTATGATGACACTATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTATGATGAGGATGAGAATCAGTCTCCCAGGAGCTTTCAGAAGAAGACCAGACATTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCTCTAGCCCTCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAATTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGGGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGGCCTTATATCAGGGCTGAGGTGGAGGATAATATTATGGTGACTTTCAGGAACCAGGCCAGCAGGCCCTACTCTTTCTATAGCAGCCTGATCTCTTATGAGGAGGATCAGAGGCAGGGGGCTGAGCCTAGGAAGAACTTTGTGAAGCCCAATGAGACTAAGACCTACTTCTGGAAGGTCCAGCACCACATGGCCCCTACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTATTTCTCTGATGTGGATCTGGAGAAGGATGTCCATTCTGGGCTGATTGGCCCCCTGCTGGTGTGCCACACTAACACTCTGAATCCTGCCCATGGCAGGCAGGTGACTGTCCAGGAGTTTGCCCTGTTCTTCACTATCTTTGATGAGACCAAGAGCTGGTACTTTACTGAGAACATGGAGAGGAACTGCAGAGCTCCTTGCAATATTCAGATGGAGGACCCCACCTTCAAGGAGAATTACAGGTTCCATGCCATTAATGGGTACATCATGGACACCCTGCCTGGCCTGGTGATGGCTCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCTCTAATGAGAATATCCACAGCATCCACTTCTCTGGGCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCTCTGTATAATCTGTACCCTGGGGTGTTTGAAACTGTGGAGATGCTGCCCTCTAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTATTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATTCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCTCTCTGTACATCTCTCAGTTCATCATCATGTACTCTCTGGATGGGAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGGAATGTGGACTCTTCTGGCATCAAGCACAACATCTTCAATCCCCCCATCATTGCTAGGTATATTAGGCTGCATCCCACCCACTACAGCATCAGGTCTACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACTCTTGCAGCATGCCCCTGGGCATGGAGTCTAAGGCCATCTCTGATGCCCAGATTACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCTCTAAGGCCAGGCTGCATCTGCAGGGGAGGAGCAATGCCTGGAGGCCTCAGGTGAACAACCCCAAGGAGTGGCTGCAGGTGGATTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTCAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACTCTGTTCTTTCAGAATGGGAAGGTGAAGGTGTTTCAGGGCAATCAGGACTCTTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATCCACCCCCAGTCTTGGGTGCATCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCTCAGGATCTGTACTGAGCGGCCGCAATAAAAGATCAGAGCTCTAGAGATCTGTGTGTTGGTTTTTTGTGTTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 14GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAGGAACCATTGCCACCTTCAGGGGGAGGCTGCTGGTGAATATTAACCAAGATCACCCCAGTTACCGGAGGAGCAAACAGGGACTAAGTTCACA205)CGCGTGGTACCGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGTTATCGTTAGTTCGTGCACCATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCATTTTTTTTTCAGGTTGGCTAGTATGCAGATCGAGCTCTCCACCTGCTTCTTTCTGTGCCTGTTGAGATTCTGCTTCAGCGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATCCACCCGTCCTTAAGCGCCATCAGCGCGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCATCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGAGGATCCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGTTTTCCTGTAACGATCGGGTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 15GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTC206)TGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGGAGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAAAGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATTTTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGTGCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTAGAGGTACCGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACGCCGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 16GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTC207)TGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGGAGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAAAGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATTTTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGTGCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTAGAGGTACCGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACGCCGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATGCCACTAATGTGTCTAACAACAGCAACACCAGCAATGACAGCAATGTGTCTCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 17GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTC208)TGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGGAGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAAAGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATTTTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGTGCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTAGAGGTACCGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAGTTTAAACGCCGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATGCCACTAATGTGTCTAACAACAGCAACACCAGCAATGACAGCAATGTGTCTCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACATGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 18GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGACGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTC209)TGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGTTTAAACGCCGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 19GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGACGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTC210)TGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGTTTAAACGCCGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATGCCACTAATGTGTCTAACAACAGCAACACCAGCAATGACAGCAATGTGTCTCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 20GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGACGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTC211)TGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGTTTAAACGCCGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATGCCACTAATGTGTCTAACAACAGCAACACCAGCAATGACAGCAATGTGTCTCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACATGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 21GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAGCGGCCGCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCT212)ATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTTTAGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTGTCGACAGAATTCCTCGAAGATCCGAAGGGGTTCAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCAGTTTAAACGCCGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCAGAAGATATTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGTCTGACCTGGGAGAGCTGCCCGTGGACGCTAGATTTCCTCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTGGAATTCACCGACCACCTGTTCAATATCGCCAAGCCTCGGCCTCCTTGGATGGGACTGCTGGGACCTACAATTCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGCCAGCCATCCTGTGTCTCTGCACGCCGTGGGAGTGTCTTATTGGAAGGCTTCTGAGGGCGCCGAGTACGACGATCAGACAAGCCAGAGAGAGAAAGAGGACGACAAGGTTTTCCCTGGCGGCAGCCACACCTATGTCTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGATCCTCTGTGCCTGACATACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAATTCTGGCCTGATCGGAGCCCTGCTCGTGTGTAGAGAAGGCAGCCTGGCCAAAGAGAAAACCCAGACACTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCTAGAGCTTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCTGGACTGATCGGCTGCCACAGAAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACAACACCTGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCTCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTCTGCCACATCAGCTCCCACCAGCACGATGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAAGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACTCTGAGATGGACGTCGTCAGATTCGACGACGATAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGATTACGCTCCTCTGGTGCTGGCCCCTGACGACAGAAGCTACAAGAGCCAGTACCTGAACAACGGCCCTCAGCGGATCGGCCGGAAGTATAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGAATTCTGGGCCCTCTGCTGTATGGCGAAGTGGGCGATACACTGCTGATCATCTTCAAGAACCAGGCCAGCAGACCCTACAACATCTACCCTCACGGCATCACCGATGTGCGGCCCCTGTATTCTAGAAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCTATCCTGCCTGGCGAGATTTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCTAGATGTCTGACACGGTACTACAGCAGCTTCGTGAACATGGAACGCGACCTGGCCAGCGGCCTGATTGGACCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTTAGCGTGTTCGATGAGAACCGGTCCTGGTATCTGACCGAGAACATCCAGCGGTTTCTGCCCAATCCTGCCGGGGTGCAACTGGAAGATCCTGAGTTCCAGGCAAGCAACATCATGCACTCCATCAATGGCTATGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAAGTGGCCTACTGGTACATCCTGAGCATTGGCGCCCAGACCGACTTCCTGTCCGTGTTCTTTAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACACTGTTCCCATTCAGCGGCGAGACAGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGTCACAACAGCGACTTCCGGAACAGAGGCATGACAGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAATGCCATCGAGCCTCGGAGCTTCAGCCAGAATCCTCCTGTGCTGAAGCGGCACCAGCGCGAGATCACCAGAACAACCCTGCAGAGCGACCAAGAGGAAATCGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAAGATTTCGACATCTACGACGAGGACGAGAATCAGAGCCCCAGAAGCTTTCAGAAAAAGACCCGGCACTACTTCATTGCCGCCGTCGAGAGACTGTGGGACTACGGCATGTCTAGCAGCCCTCACGTGCTGAGAAATAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAAGAGTTCACCGACGGCAGCTTCACCCAGCCACTGTATAGAGGCGAGCTGAACGAGCATCTGGGCCTGCTGGGCCCTTATATCAGAGCCGAAGTGGAAGATAACATCATGGTCACCTTCCGGAATCAGGCTAGCCGGCCTTACAGCTTCTACAGCTCCCTGATCAGCTACGAAGAGGACCAGAGACAGGGCGCTGAGCCCAGAAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTTTGGAAGGTGCAGCACCACATGGCCCCTACAAAGGACGAGTTCGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGATCTGGAAAAGGACGTGCACAGCGGGCTCATCGGACCACTGCTTGTGTGCCACACCAACACACTGAACCCCGCTCACGGCAGACAAGTGACAGTGCAAGAGTTCGCCCTGTTCTTCACCATCTTCGACGAAACAAAGAGCTGGTACTTCACCGAGAATATGGAACGGAACTGCAGAGCCCCTTGCAACATCCAGATGGAAGATCCCACCTTCAAAGAGAACTACCGGTTCCACGCCATCAACGGCTACATCATGGACACACTGCCCGGCCTGGTTATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGTCCATGGGCTCCAACGAGAATATCCACAGCATCCACTTCAGCGGCCACGTGTTCACCGTGCGGAAAAAAGAAGAGTACAAAATGGCCCTGTACAATCTGTACCCTGGGGTGTTCGAAACCGTGGAAATGCTGCCTTCCAAGGCCGGCATTTGGAGAGTGGAATGTCTGATTGGAGAGCACCTCCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGTCAGACCCCTCTCGGCATGGCCTCTGGACACATCAGAGACTTCCAGATCACCGCCTCTGGCCAGTACGGACAGTGGGCTCCTAAACTGGCTCGGCTGCACTACAGCGGCAGCATCAATGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCTCCCATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGAGGCAACAGCACCGGCACACTCATGGTGTTCTTCGGCAACGTGGACTCCAGCGGCATTAAGCACAACATCTTCAACCCTCCAATCATTGCCCGGTACATCCGGCTGCACCCCACACACTACAGCATCCGGTCTACCCTGAGAATGGAACTGATGGGCTGCGACCTGAACAGCTGCTCTATGCCCCTCGGAATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCCAGCAGCTACTTCACCAACATGTTCGCCACTTGGAGCCCCTCCAAGGCTAGACTGCATCTGCAGGGCAGAAGCAACGCTTGGAGGCCCCAAGTGAACAACCCCAAAGAGTGGCTGCAGGTTGACTTTCAAAAGACCATGAAAGTGACCGGCGTGACCACACAGGGCGTCAAGTCTCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCTCCAGCAGCCAGGACGGCCATCAGTGGACCCTGTTTTTCCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAATCAGGACAGCTTCACACCCGTGGTCAATTCTCTGGACCCTCCACTGCTGACCAGATACCTGCGGATTCACCCTCAGTCTTGGGTGCACCAGATCGCTCTGCGGATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTCTACTAGTTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACATGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 22GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGAGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGG213)TGAGTCAACAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCCTGCAGCCCTAGACAATTGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGCTTCCGTTTAAACGCCGCCACCATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAATGCCACTAATGTGTCTAACAACAGCAACACCAGCAATGACAGCAATGTGTCTCCCCCAGTGCTGAAGAGGCACCAGAGGGAGATCACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATTGACTATGATGACACCATCTCTGTGGAGATGAAGAAGGAGGACTTTGACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACATGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGATCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 23GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGACGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTC214)TGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGTTTAAACGCCGCCACCATGCAAATTGAGTTAAGTACCTGTTTTTTCCTGTGCCTGTTGAGATTTTGCTTCTCTGCTACCAGAAGGTATTACCTGGGAGCTGTGGAGCTGTCATGGGACTACATGCAGTCTGACCTGGGAGAGCTCCCTGTTGATGCTAGGTTTCCCCCTAGGGTGCCAAAGTCCTTTCCTTTTAACACCTCAGTGGTGTACAAGAAGACACTGTTTGTGGAGTTTACTGACCACCTCTTCAATATTGCTAAGCCAAGACCCCCTTGGATGGGTCTCTTGGGCCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTGATTACCCTCAAGAATATGGCTTCTCATCCTGTTAGTCTGCATGCAGTAGGGGTCAGCTATTGGAAGGCCAGTGAAGGAGCTGAGTATGATGACCAGACCAGCCAGAGAGAGAAAGAGGATGACAAGGTGTTCCCTGGGGGATCACACACCTATGTGTGGCAGGTGTTGAAGGAAAATGGACCCATGGCCTCTGACCCACTCTGCCTGACTTACTCCTACCTGTCTCATGTGGACCTGGTCAAAGATCTGAACTCTGGCCTGATTGGGGCACTGTTGGTGTGCAGAGAAGGGTCTCTTGCCAAGGAGAAGACTCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAAGGCAAGTCTTGGCATTCTGAGACTAAGAACAGCCTGATGCAAGATAGAGATGCTGCATCTGCCAGGGCCTGGCCTAAGATGCACACTGTGAATGGATATGTGAATAGGTCCTTGCCTGGCCTGATTGGATGCCATAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTTCACTCTATTTTTCTGGAGGGCCATACTTTCCTTGTGAGGAATCATAGACAGGCCTCTCTGGAGATTAGCCCTATTACATTTCTGACTGCTCAGACCCTCCTCATGGACTTGGGGCAGTTTCTGTTAAGCTGCCACATCAGCAGCCATCAACATGATGGTATGGAAGCCTATGTCAAAGTGGATAGCTGCCCTGAAGAACCACAGCTTAGGATGAAGAACAATGAGGAGGCAGAGGACTATGATGATGATCTGACTGATTCTGAAATGGATGTGGTGAGATTTGATGATGATAATAGCCCTTCCTTTATCCAGATCAGGTCAGTGGCCAAGAAGCACCCAAAGACCTGGGTGCATTATATTGCTGCTGAGGAGGAGGACTGGGACTATGCCCCTCTGGTGCTGGCCCCTGATGATAGAAGCTATAAGTCTCAATACTTGAATAATGGCCCACAGAGGATTGGGAGAAAGTACAAAAAGGTGAGATTTATGGCCTACACTGATGAGACTTTCAAGACCAGAGAGGCCATCCAGCATGAATCAGGCATTTTGGGTCCCCTGCTGTATGGAGAGGTGGGGGATACCCTGCTGATCATCTTTAAGAACCAGGCCAGCAGACCTTACAACATTTACCCTCATGGGATCACAGATGTGAGGCCACTCTACTCTAGGAGATTGCCCAAGGGAGTCAAGCATCTTAAGGACTTTCCAATCCTGCCTGGGGAGATCTTCAAGTACAAATGGACAGTGACAGTTGAGGATGGACCTACTAAGAGTGACCCCAGGTGTCTGACCAGATATTACTCTAGCTTTGTCAATATGGAAAGAGACCTGGCTTCAGGCTTGATAGGCCCTCTTCTCATCTGCTACAAAGAGTCTGTGGACCAGAGAGGAAATCAGATTATGTCTGACAAGAGAAATGTTATTCTGTTTTCAGTCTTTGATGAGAACAGAAGTTGGTACTTGACAGAGAACATTCAGAGATTTCTGCCTAACCCTGCAGGAGTGCAGCTTGAGGACCCTGAGTTTCAGGCTTCCAATATCATGCATTCTATCAATGGCTATGTGTTTGATAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTTGCCTATTGGTACATCCTGTCTATTGGGGCCCAAACTGATTTCTTGTCTGTGTTTTTCAGTGGATATACCTTTAAGCATAAGATGGTGTATGAAGATACCTTGACACTTTTCCCTTTCTCAGGAGAGACTGTCTTCATGTCTATGGAGAACCCTGGACTGTGGATCCTGGGATGCCACAACTCAGACTTCAGAAACAGGGGTATGACAGCTCTGCTTAAGGTCTCCTCATGTGACAAAAATACAGGAGACTACTATGAAGATTCCTATGAGGACATCTCTGCTTACCTGCTCAGCAAAAACAATGCCATTGAGCCCAGAAGCTTCAGCCAGAATTCCAGACATCCCAGTACTAGGCAGAAGCAGTTTAATGCCACCACCATCCCAGAAAATGACATTGAGAAGACAGACCCTTGGTTTGCCCATAGAACCCCAATGCCCAAGATTCAGAATGTGAGCAGCTCAGACTTGCTGATGCTGCTGAGGCAGTCTCCTACTCCCCATGGCCTGTCCCTCTCAGATCTGCAGGAAGCCAAGTATGAGACATTTTCTGATGACCCCTCCCCAGGGGCTATTGACAGTAACAACAGCCTGTCTGAAATGACCCACTTCAGACCTCAGCTCCACCACAGTGGGGATATGGTGTTTACCCCAGAGTCAGGCCTGCAGCTGAGACTGAATGAGAAGCTGGGAACCACAGCTGCCACAGAACTGAAGAAGCTGGATTTCAAGGTGAGCTCTACTTCAAATAATCTGATTTCCACTATCCCATCTGACAATTTGGCAGCTGGCACTGACAATACAAGCTCTCTGGGCCCACCTAGCATGCCAGTGCATTATGACAGCCAGTTGGACACCACTCTTTTTGGAAAGAAATCCAGTCCCCTGACAGAGTCAGGAGGACCCCTTTCTCTGTCTGAGGAGAACAATGATAGTAAGCTTCTGGAGTCAGGCTTGATGAACTCCCAGGAGAGTTCTTGGGGCAAGAATGTGAGCTCTAGGGAGATCACCAGAACCACTCTGCAGTCTGACCAAGAAGAGATTGATTATGATGACACTATCTCTGTTGAGATGAAGAAGGAGGACTTTGATATCTATGATGAGGATGAGAACCAGTCTCCAAGAAGTTTCCAGAAGAAGACCAGGCACTACTTTATTGCTGCTGTGGAGAGACTGTGGGACTATGGAATGTCCTCCTCCCCTCATGTCCTGAGAAATAGAGCTCAGTCAGGATCAGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAATTCACAGATGGAAGTTTTACCCAGCCACTGTATAGAGGTGAACTGAATGAACATCTGGGTCTGCTGGGGCCCTATATCAGGGCTGAGGTGGAGGACAATATTATGGTGACCTTTAGAAACCAGGCCTCAAGGCCCTACAGCTTCTACTCTTCCCTCATTTCATATGAGGAAGACCAGAGACAGGGAGCAGAGCCTAGAAAGAACTTTGTCAAGCCCAATGAAACCAAAACCTATTTCTGGAAGGTGCAGCACCACATGGCACCCACCAAAGATGAGTTTGACTGCAAAGCCTGGGCCTATTTCTCAGATGTGGATCTGGAGAAGGATGTCCATTCAGGTCTGATTGGACCACTCCTGGTGTGCCACACCAACACACTGAATCCAGCTCATGGCAGACAGGTGACAGTCCAGGAGTTTGCCCTGTTTTTCACTATTTTTGATGAGACCAAGAGCTGGTATTTTACAGAGAATATGGAAAGAAACTGCAGAGCCCCTTGTAATATCCAGATGGAGGACCCAACCTTTAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATTATGGATACCCTGCCTGGCCTGGTTATGGCCCAGGATCAGAGGATCAGGTGGTATCTGCTGTCAATGGGCTCCAATGAGAACATTCATAGTATTCACTTTTCAGGACATGTGTTCACAGTGAGAAAGAAGGAGGAGTATAAGATGGCCCTCTATAATCTCTACCCAGGGGTGTTTGAGACAGTTGAGATGCTGCCATCTAAGGCAGGCATCTGGAGAGTGGAATGCCTCATTGGAGAGCACCTGCATGCTGGCATGTCCACCCTGTTTCTGGTGTACTCCAACAAATGTCAGACCCCTCTTGGTATGGCCTCTGGCCACATTAGGGATTTCCAGATTACTGCCAGTGGTCAGTATGGGCAGTGGGCCCCTAAGCTGGCCAGACTGCACTATTCTGGGAGCATCAATGCCTGGTCCACCAAGGAACCTTTTTCTTGGATTAAAGTGGATCTGCTGGCCCCAATGATCATCCATGGGATCAAGACCCAGGGAGCTAGGCAGAAGTTCAGCAGCCTGTACATTAGTCAGTTCATCATTATGTACAGTCTGGATGGCAAAAAGTGGCAGACATACAGGGGCAATTCCACTGGAACCCTGATGGTGTTCTTTGGAAATGTGGATAGCAGTGGTATCAAGCACAACATTTTCAACCCTCCCATCATTGCCAGGTACATCAGACTGCATCCAACCCACTACAGCATTAGGAGCACCCTTAGGATGGAGCTCATGGGGTGTGACCTGAACTCTTGCTCTATGCCACTTGGAATGGAGAGCAAGGCCATCTCTGATGCCCAGATTACTGCTAGTTCCTACTTCACCAATATGTTTGCCACCTGGTCACCCAGCAAGGCCAGGCTGCACCTGCAGGGAAGGTCCAATGCTTGGAGGCCTCAGGTGAACAATCCAAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACTATGAAGGTGACAGGAGTGACCACTCAGGGAGTGAAGAGCCTGCTGACCTCCATGTATGTGAAGGAGTTTCTGATCTCCAGCTCCCAGGATGGCCACCAGTGGACCCTGTTCTTTCAGAATGGCAAGGTGAAGGTGTTTCAGGGGAATCAGGACTCCTTCACCCCAGTGGTGAATTCCCTGGATCCCCCTCTGCTGACCAGATACCTGAGAATCCACCCCCAGTCTTGGGTGCACCAGATTGCCCTTAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCceDNAFVIII-GGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAvector 24GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGC(SEQ ID NO:TCTAGACGGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACGCGTGGTACCGTCTGTC215)TGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTGAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGTTTAAACGCCGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTGCCAAAATCTTTTCCATTCAACACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTTTCAACATCGCTAAGCCAAGGCCACCCTGGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTCAGTCTTCATGCTGTTGGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGGGAGAAAGAAGATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCACTGTGCCTTACCTACTCATATCTTTCTCATGTGGACCTGGTAAAAGACTTGAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAAGGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAATTTATACTACTTTTTGCTGTATTTGATGAAGGGAAAAGTTGGCACTCAGAAACAAAGAACTCCTTGATGCAGGATAGGGATGCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGGTTATGTAAACAGGTCTCTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATTGGCATGTGATTGGAATGGGCACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTCACACATTTCTTGTGAGGAACCATCGCCAGGCGTCCTTGGAAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTCTTGATGGACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGCATGGAAGCTTATGTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAAGAAGCGGAAGACTATGATGATGATCTTACTGATTCTGAAATGGATGTGGTCAGGTTTGATGATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCCAAGAAGCATCCTAAAACTTGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCCCCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAAGTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCTATTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTGTTGATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCGTCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTTCCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAATCAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAGAACCGAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAGTTCCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGTTTGCAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGCATTGGAGCACAGACTGACTTCCTTTCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACACACTCACCCTATTCCCATTCTCAGGAGAAACTGTCTTCATGTCGATGGAAAACCCAGGTCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAACAGAGGCATGACCGCCTTACTGAAGGTTTCTAGTTGTGACAAGAACACTGGTGATTATTACGAGGACAGTTATGAAGATATTTCAGCATACTTGCTGAGTAAAAACAATGCCATTGAACCAAGAAGCTTCTCCCAGAATTCAAGACACCCTAGCACTAGGCAAAAGCAATTTAATGCCACCACAATTCCAGAAAATGACATAGAGAAGACTGACCCTTGGTTTGCACACAGAACACCTATGCCTAAAATACAAAATGTCTCCTCTAGTGATTTGTTGATGCTCTTGCGACAGAGTCCTACTCCACATGGGCTATCCTTATCTGATCTCCAAGAAGCCAAATATGAGACTTTTTCTGATGATCCATCACCTGGAGCAATAGACAGTAATAACAGCCTGTCTGAAATGACACACTTCAGGCCACAGCTCCATCACAGTGGGGACATGGTATTTACCCCTGAGTCAGGCCTCCAATTAAGATTAAATGAGAAACTGGGGACAACTGCAGCAACAGAGTTGAAGAAACTTGATTTCAAAGTTTCTAGTACATCAAATAATCTGATTTCAACAATTCCATCAGACAATTTGGCAGCAGGTACTGATAATACAAGTTCCTTAGGACCCCCAAGTATGCCAGTTCATTATGATAGTCAATTAGATACCACTCTATTTGGCAAAAAGTCATCTCCCCTTACTGAGTCTGGTGGACCTCTGAGCTTGAGTGAAGAAAATAATGATTCAAAGTTGTTAGAATCAGGTTTAATGAATAGCCAAGAAAGTTCATGGGGAAAAAATGTATCGTCAACAGAGAGTGGTAGGTTATTTAAAGGGAAAAGAGCTCATGGACCTGCTTTGTTGACTAAAGATAATGCCTTATTCAAAGTTAGCATCTCTTTGTTAAAGACAAACAAAACTTCCAATAATTCAGCAACTAATAGAAAGACTCACATTGATGGCCCATCATTATTAATTGAGAATAGTCCATCAGTCTGGCAAAATATATTAGAAAGTGACACTGAGTTTAAAAAAGTGACACCTTTGATTCATGACAGAATGCTTATGGACAAAAATGCTACAGCTTTGAGGCTAAATCATATGTCAAATAAAACTACTTCATCAAAAAACATGGAAATGGTCCAACAGAAAAAAGAGGGCCCCATTCCACCAGATGCACAAAATCCAGATATGTCGTTCTTTAAGATGCTATTCTTGCCAGAATCAGCAAGGTGGATACAAAGGACTCATGGAAAGAACTCTCTGAACTCTGGGCAAGGCCCCAGTCCAAAGCAATTAGTATCCTTAGGACCAGAAAAATCTGTGGAAGGTCAGAATTTCTTGTCTGAGAAAAACAAAGTGGTAGTAGGAAAGGGTGAATTTACAAAGGACGTAGGACTCAAAGAGATGGTTTTTCCAAGCAGCAGAAACCTATTTCTTACTAACTTGGATAATTTACATGAAAATAATACACACAATCAAGAAAAAAAAATTCAGGAAGAAATAGAAAAGAAGGAAACATTAATCCAAGAGAATGTAGTTTTGCCTCAGATACATACAGTGACTGGCACTAAGAATTTCATGAAGAACCTTTTCTTACTGAGCACTAGGCAAAATGTAGAAGGTTCATATGACGGGGCATATGCTCCAGTACTTCAAGATTTTAGGTCATTAAATGATTCAACAAATAGAACAAAGAAACACACAGCTCATTTCTCAAAAAAAGGGGAGGAAGAAAACTTGGAAGGCTTGGGAAATCAAACCAAGCAAATTGTAGAGAAATATGCATGCACCACAAGGATATCTCCTAATACAAGCCAGCAGAATTTTGTCACGCAACGTAGTAAGAGAGCTTTGAAACAATTCAGACTCCCACTAGAAGAAACAGAACTTGAAAAAAGGATAATTGTGGATGACACCTCAACCCAGTGGTCCAAAAACATGAAACATTTGACCCCGAGCACCCTCACACAGATAGACTACAATGAGAAGGAGAAAGGGGCCATTACTCAGTCTCCCTTATCAGATTGCCTTACGAGGAGTCATAGCATCCCTCAAGCAAATAGATCTCCATTACCCATTGCAAAGGTATCATCATTTCCATCTATTAGACCTATATATCTGACCAGGGTCCTATTCCAAGACAACTCTTCTCATCTTCCAGCAGCATCTTATAGAAAGAAAGATTCTGGGGTCCAAGAAAGCAGTCATTTCTTACAAGGAGCCAAAAAAAATAACCTTTCTTTAGCCATTCTAACCTTGGAGATGACTGGTGATCAAAGAGAGGTTGGCTCCCTGGGGACAAGTGCCACAAATTCAGTCACATACAAGAAAGTTGAGAACACTGTTCTCCCGAAACCAGACTTGCCCAAAACATCTGGCAAAGTTGAATTGCTTCCAAAAGTTCACATTTATCAGAAGGACCTATTCCCTACGGAAACTAGCAATGGGTCTCCTGGCCATCTGGATCTCGTGGAAGGGAGCCTTCTTCAGGGAACAGAGGGAGCGATTAAGTGGAATGAAGCAAACAGACCTGGAAAAGTTCCCTTTCTGAGAGTAGCAACAGAAAGCTCTGCAAAGACTCCCTCCAAGCTATTGGATCCTCTTGCTTGGGATAACCACTATGGTACTCAGATACCAAAAGAAGAGTGGAAATCCCAAGAGAAGTCACCAGAAAAAACAGCTTTTAAGAAAAAGGATACCATTTTGTCCCTGAACGCTTGTGAAAGCAATCATGCAATAGCAGCAATAAATGAGGGACAAAATAAGCCCGAAATAGAAGTCACCTGGGCAAAGCAAGGTAGGACTGAAAGGCTGTGCTCTCAAAACCCACCAGTCTTGAAACGCCATCAACGGGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGACTATGATGATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACACGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGGATTATGGGATGAGTAGCTCCCCACATGTTCTAAGAAACAGGGCTCAGAGTGGCAGTGTCCCTCAGTTCAAGAAAGTTGTTTTCCAGGAATTTACTGATGGCTCCTTTACTCAGCCCTTATACCGTGGAGAACTAAATGAACATTTGGGACTCCTGGGGCCATATATAAGAGCAGAAGTTGAAGATAATATCATGGTAACTTTCAGAAATCAGGCCTCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGGAAGATCAGAGGCAAGGAGCAGAACCTAGAAAAAACTTTGTCAAGCCTAATGAAACCAAAACTTACTTTTGGAAAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGGGCTTATTTCTCTGATGTTGACCTGGAAAAAGATGTGCACTCAGGCCTGATTGGACCCCTTCTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGGAATTTGCTCTGTTTTTCACCATCTTTGATGAGACCAAAAGCTGGTACTTCACTGAAAATATGGAAAGAAACTGCAGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAATTATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGGCTTAGTAATGGCTCAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCTATTCATTTCAGTGGACATGTGTTCACTGTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTATCCAGGTGTTTTTGAGACAGTGGAAATGTTACCATCCAAAGCTGGAATTTGGCGGGTGGAATGCCTTATTGGCGAGCATCTACATGCTGGGATGAGCACACTTTTTCTGGTGTACAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTTCAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTGTTGGCACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGCCTCTACATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATAAAACACAATATTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCATTATAGCATTCGCAGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGCATGCCATTGGGAATGGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTACTTTACCAATATGTTTGCCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGGAGTAATGCCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGGAGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTTCAGAATGGCAAAGTAAAGGTTTTTCAGGGAAATCAAGACTCCTTCACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGACTCGCTACCTTCGAATTCACCCCCAGAGTTGGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTCTACTGATTAATTAAGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCTCGAGGCATGCGGTACCAAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGATCACTGATATCGCCTAGGAGATCCGAACCAGATAAGTGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTACGACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCTTAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAAGCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAAACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATCAGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAGCAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGGCGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTCGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCTAGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGGATCTCGGTCCGAAACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCC ceDNAFVIII-AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACATAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATvector 25AGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATA(SEQ ID NO:CGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCCTAGGCTCAAGCAGTGATCAGATCCAGACATGATAAGA216)TACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCTCTAGTACTTCTCGACAAGCTTGTCGAGACTGCAGGCTCTAGAGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCATATGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAACAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCCTGCAGCCCTAGACAATTGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGCTTCCGTTTAAACAGCCACCATGCAGATAGAATTATCTACTTGTTTTTTTCTTTGTCTGCTCAGGTTCTGCTTCTCTGCTACAAGGAGGTATTACTTAGGTGCTGTAGAGTTATCTTGGGACTACATGCAGTCTGATCTTGGGGAGCTCCCTGTGGATGCAAGATTTCCCCCTAGAGTACCTAAAAGTTTCCCTTTCAACACAAGTGTGGTGTATAAGAAGACTCTGTTTGTTGAGTTCACAGATCACCTTTTCAATATTGCCAAGCCCAGACCCCCCTGGATGGGTCTACTGGGGCCAACAATTCAAGCAGAGGTATATGATACAGTGGTGATCACATTAAAGAACATGGCATCCCACCCTGTAAGCCTGCATGCTGTTGGTGTGTCCTATTGGAAAGCAAGTGAAGGTGCTGAATATGATGATCAAACATCCCAAAGAGAGAAGGAAGATGACAAAGTCTTCCCAGGTGGCTCTCACACCTATGTGTGGCAAGTGCTCAAGGAAAATGGGCCTATGGCTAGTGATCCATTGTGCCTCACATATAGCTACCTGTCCCATGTGGACTTAGTTAAAGATCTGAACTCAGGTCTGATTGGTGCCCTGCTGGTCTGTAGGGAAGGATCACTAGCTAAAGAAAAAACCCAGACTTTACACAAGTTCATCTTATTATTTGCTGTTTTTGATGAGGGAAAGAGCTGGCACTCAGAGACTAAGAATTCACTCATGCAGGATAGAGATGCAGCTTCTGCTAGAGCATGGCCAAAAATGCATACTGTGAATGGGTATGTTAACAGGAGTCTGCCTGGCCTCATAGGCTGCCATAGAAAATCAGTGTACTGGCATGTCATAGGTATGGGCACTACTCCTGAGGTGCATTCCATTTTTTTGGAGGGGCATACTTTCCTTGTAAGAAACCACAGGCAGGCTTCATTGGAAATTAGTCCAATAACCTTTCTCACAGCCCAGACTCTGCTAATGGACTTGGGGCAGTTTTTATTATTTTGTCACATTAGCTCTCATCAGCATGATGGAATGGAAGCCTATGTTAAGGTTGATTCCTGCCCAGAAGAGCCCCAGTTGAGAATGAAGAACAATGAGGAAGCAGAAGACTATGATGATGATCTAACAGACTCAGAAATGGATGTTGTGAGATTTGATGATGATAACTCCCCAAGTTTTATTCAGATCAGGTCAGTGGCCAAGAAACATCCAAAAACATGGGTTCATTATATTGCAGCAGAAGAAGAAGACTGGGATTATGCACCCTTGGTATTAGCCCCTGATGACAGAAGCTATAAGTCACAATATCTCAACAATGGACCCCAGAGAATTGGCAGAAAGTACAAAAAAGTCAGGTTTATGGCTTATACTGATGAGACTTTCAAAACTAGAGAGGCTATTCAACATGAATCTGGCATCCTAGGTCCTTTGTTATATGGGGAAGTAGGGGACACCTTATTAATTATATTTAAAAACCAGGCCAGTAGGCCTTACAACATTTACCCCCACGGTATAACAGATGTCAGACCACTGTACTCAAGGAGGCTACCAAAAGGGGTGAAGCACCTAAAAGACTTCCCCATCCTACCTGGTGAGATTTTCAAGTATAAGTGGACAGTGACTGTAGAAGATGGTCCAACCAAATCAGATCCCAGATGCCTGACAAGGTACTACAGTAGCTTTGTAAATATGGAGAGAGACCTGGCCTCAGGGTTAATAGGGCCCCTACTGATCTGCTACAAGGAGAGTGTAGACCAAAGAGGCAACCAAATTATGTCAGACAAGAGAAATGTCATCCTCTTTAGTGTTTTTGATGAGAATAGGTCCTGGTACCTGACAGAAAATATCCAAAGATTCCTTCCAAATCCAGCAGGGGTACAACTGGAGGACCCTGAGTTCCAGGCCTCCAATATCATGCACAGCATCAATGGTTATGTGTTTGACAGTTTGCAACTTTCAGTGTGCCTTCATGAGGTTGCATACTGGTATATACTTTCTATAGGGGCCCAGACAGACTTCTTGTCAGTTTTCTTTTCAGGCTATACTTTCAAGCATAAGATGGTATATGAAGATACCCTGACTCTTTTTCCCTTTAGTGGTGAGACAGTTTTCATGTCAATGGAAAACCCAGGGCTTTGGATCTTGGGATGCCACAACTCTGACTTTAGAAACAGAGGCATGACTGCCCTTTTAAAGGTGTCCTCTTGTGATAAGAATACTGGAGACTACTATGAGGACAGCTATGAGGACATTTCAGCCTATCTGCTGTCAAAAAATAATGCTATAGAGCCAAGGTCATTTTCTCAAAATCCTCCTGTCCTGAAGAGGCACCAAAGAGAGATAACCAGGACTACCTTACAGTCTGACCAGGAGGAAATTGATTATGATGACACAATAAGTGTGGAGATGAAAAAGGAGGATTTTGATATTTATGATGAGGATGAAAACCAATCTCCAAGATCTTTCCAGAAGAAGACTAGACACTATTTCATTGCTGCAGTGGAAAGACTGTGGGACTATGGAATGTCCTCCAGCCCTCATGTTCTCAGGAATAGGGCTCAAAGTGGTTCTGTGCCACAGTTCAAGAAGGTGGTGTTTCAGGAATTCACAGATGGGTCCTTTACTCAACCACTGTATAGGGGAGAACTCAATGAACATCTAGGATTACTAGGTCCCTACATTAGAGCTGAGGTGGAGGACAATATCATGGTCACTTTCAGGAACCAGGCATCTAGACCATACTCTTTTTACAGCTCCTTGATATCTTATGAAGAGGACCAGAGACAGGGTGCAGAACCCAGGAAGAACTTTGTGAAACCCAATGAGACCAAAACATACTTCTGGAAGGTCCAGCATCATATGGCTCCTACCAAGGATGAATTTGACTGCAAAGCCTGGGCTTACTTCTCAGATGTGGATTTGGAGAAAGATGTGCACAGTGGCTTGATTGGGCCTCTTTTGGTATGTCATACCAACACCTTAAACCCTGCTCATGGAAGACAAGTAACAGTGCAAGAGTTTGCCCTCTTTTTCACTATATTTGATGAGACAAAAAGTTGGTACTTTACTGAGAATATGGAGAGGAACTGCAGGGCTCCCTGCAATATACAGATGGAGGATCCTACTTTCAAGGAAAACTATAGGTTTCATGCCATTAATGGCTATATCATGGATACATTGCCTGGCTTAGTTATGGCCCAGGATCAGAGAATCAGATGGTACCTCCTTAGTATGGGAAGCAATGAAAACATACATTCTATTCACTTCAGTGGACATGTCTTTACTGTGAGAAAAAAGGAAGAGTACAAGATGGCACTGTACAATCTGTACCCTGGAGTGTTTGAGACAGTTGAGATGCTGCCCAGCAAGGCTGGGATCTGGAGGGTAGAATGCCTGATTGGAGAACACTTACATGCAGGTATGTCCACTCTATTCTTGGTCTATTCCAACAAGTGTCAGACCCCCCTAGGCATGGCATCTGGCCATATTAGAGATTTCCAGATAACTGCTTCTGGTCAGTATGGCCAGTGGGCCCCCAAGCTTGCTAGACTTCACTACAGTGGTTCAATAAATGCCTGGTCCACCAAGGAGCCCTTCAGCTGGATTAAAGTGGACTTGCTAGCCCCCATGATCATTCATGGGATAAAAACACAGGGTGCCAGACAGAAATTTTCATCTCTGTACATCAGCCAGTTTATCATCATGTACAGTCTGGATGGCAAAAAATGGCAGACCTACAGAGGTAACTCTACAGGTACACTAATGGTATTTTTTGGCAATGTAGATTCATCAGGTATCAAGCATAATATCTTTAATCCTCCCATTATTGCCAGATACATCAGGTTGCACCCAACTCATTACAGTATAAGATCCACTTTAAGGATGGAGCTTATGGGGTGTGACCTTAATAGCTGCAGTATGCCCTTGGGAATGGAAAGTAAGGCCATATCAGATGCCCAGATTACTGCAAGCAGCTACTTTACAAACATGTTTGCAACCTGGTCTCCTAGTAAAGCCAGGCTGCACTTGCAGGGTAGGAGCAATGCATGGAGACCCCAGGTCAATAACCCAAAAGAGTGGCTTCAAGTTGACTTTCAAAAGACAATGAAGGTAACAGGGGTAACCACCCAAGGGGTAAAGAGTCTGCTGACATCTATGTATGTAAAAGAATTCCTCATCTCAAGTAGTCAAGATGGGCACCAGTGGACACTGTTCTTCCAGAATGGAAAAGTTAAGGTGTTCCAAGGTAATCAAGACAGCTTCACTCCAGTGGTGAACAGTCTGGATCCACCACTCCTAACCAGGTACCTGAGGATCCACCCTCAGTCCTGGGTCCACCAGATAGCTCTGAGAATGGAAGTGCTGGGCTGTGAAGCTCAGGACCTATACTGATTAATTAATAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTGCAATGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACACAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTACTTAAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCGCTAGCAGCTGATGCATAGCATGCGGTACCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACTACGGGTACGTAGGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACGGCCGAGGTCTACCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGCAGGGCATCCATTTATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCTGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCAAATAAGCGTTGATATTCAGTCAATTACAAACATTAATAACGAAGAGATGACAGAAAAATTTTCATTCTGTGACAGAGAA ceDNAFVIII-AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAACATAACAGGAAGAAAAATGCCCCGCTGTGGGCGGACAAAATvector 26AGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTATTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATA(SEQ ID NO:CGAAAATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTTCGGATCTCCTAGGCTCAAGCAGTGATCAGATCCAGACATGATAAGA217)TACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCTCTAGTACTTCTCGACAAGCTTGTCGAGACTGCAGGCTCTAGAGGCCGGCCCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCATATGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAACAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCCTGCAGCCCTAGACAATTGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGCTTCCGTTTAAACAGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTGCCAAAATCTTTTCCATTCAACACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTTTCAACATCGCTAAGCCAAGGCCACCCTGGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTCAGTCTTCATGCTGTTGGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGGGAGAAAGAAGATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCACTGTGCCTTACCTACTCATATCTTTCTCATGTGGACCTGGTAAAAGACTTGAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAAGGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAATTTATACTACTTTTTGCTGTATTTGATGAAGGGAAAAGTTGGCACTCAGAAACAAAGAACTCCTTGATGCAGGATAGGGATGCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGGTTATGTAAACAGGTCTCTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATTGGCATGTGATTGGAATGGGCACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTCACACATTTCTTGTGAGGAACCATCGCCAGGCGTCCTTGGAAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTCTTGATGGACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGCATGGAAGCTTATGTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAAGAAGCGGAAGACTATGATGATGATCTTACTGATTCTGAAATGGATGTGGTCAGGTTTGATGATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCCAAGAAGCATCCTAAAACTTGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCCCCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAAGTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCTATTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTGTTGATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCGTCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTTCCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAATCAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAGAACCGAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAGTTCCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGTTTGCAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGCATTGGAGCACAGACTGACTTCCTTTCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACACACTCACCCTATTCCCATTCTCAGGAGAAACTGTCTTCATGTCGATGGAAAACCCAGGTCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAACAGAGGCATGACCGCCTTACTGAAGGTTTCTAGTTGTGACAAGAACACTGGTGATTATTACGAGGACAGTTATGAAGATATTTCAGCATACTTGCTGAGTAAAAACAATGCCATTGAACCAAGAAGCTTCTCCCAGAATTCAAGACACCCTAGCACTAGGCAAAAGCAATTTAATGCCACCACAATTCCAGAAAATGACATAGAGAAGACTGACCCTTGGTTTGCACACAGAACACCTATGCCTAAAATACAAAATGTCTCCTCTAGTGATTTGTTGATGCTCTTGCGACAGAGTCCTACTCCACATGGGCTATCCTTATCTGATCTCCAAGAAGCCAAATATGAGACTTTTTCTGATGATCCATCACCTGGAGCAATAGACAGTAATAACAGCCTGTCTGAAATGACACACTTCAGGCCACAGCTCCATCACAGTGGGGACATGGTATTTACCCCTGAGTCAGGCCTCCAATTAAGATTAAATGAGAAACTGGGGACAACTGCAGCAACAGAGTTGAAGAAACTTGATTTCAAAGTTTCTAGTACATCAAATAATCTGATTTCAACAATTCCATCAGACAATTTGGCAGCAGGTACTGATAATACAAGTTCCTTAGGACCCCCAAGTATGCCAGTTCATTATGATAGTCAATTAGATACCACTCTATTTGGCAAAAAGTCATCTCCCCTTACTGAGTCTGGTGGACCTCTGAGCTTGAGTGAAGAAAATAATGATTCAAAGTTGTTAGAATCAGGTTTAATGAATAGCCAAGAAAGTTCATGGGGAAAAAATGTATCGTCAACAGAGAGTGGTAGGTTATTTAAAGGGAAAAGAGCTCATGGACCTGCTTTGTTGACTAAAGATAATGCCTTATTCAAAGTTAGCATCTCTTTGTTAAAGACAAACAAAACTTCCAATAATTCAGCAACTAATAGAAAGACTCACATTGATGGCCCATCATTATTAATTGAGAATAGTCCATCAGTCTGGCAAAATATATTAGAAAGTGACACTGAGTTTAAAAAAGTGACACCTTTGATTCATGACAGAATGCTTATGGACAAAAATGCTACAGCTTTGAGGCTAAATCATATGTCAAATAAAACTACTTCATCAAAAAACATGGAAATGGTCCAACAGAAAAAAGAGGGCCCCATTCCACCAGATGCACAAAATCCAGATATGTCGTTCTTTAAGATGCTATTCTTGCCAGAATCAGCAAGGTGGATACAAAGGACTCATGGAAAGAACTCTCTGAACTCTGGGCAAGGCCCCAGTCCAAAGCAATTAGTATCCTTAGGACCAGAAAAATCTGTGGAAGGTCAGAATTTCTTGTCTGAGAAAAACAAAGTGGTAGTAGGAAAGGGTGAATTTACAAAGGACGTAGGACTCAAAGAGATGGTTTTTCCAAGCAGCAGAAACCTATTTCTTACTAACTTGGATAATTTACATGAAAATAATACACACAATCAAGAAAAAAAAATTCAGGAAGAAATAGAAAAGAAGGAAACATTAATCCAAGAGAATGTAGTTTTGCCTCAGATACATACAGTGACTGGCACTAAGAATTTCATGAAGAACCTTTTCTTACTGAGCACTAGGCAAAATGTAGAAGGTTCATATGACGGGGCATATGCTCCAGTACTTCAAGATTTTAGGTCATTAAATGATTCAACAAATAGAACAAAGAAACACACAGCTCATTTCTCAAAAAAAGGGGAGGAAGAAAACTTGGAAGGCTTGGGAAATCAAACCAAGCAAATTGTAGAGAAATATGCATGCACCACAAGGATATCTCCTAATACAAGCCAGCAGAATTTTGTCACGCAACGTAGTAAGAGAGCTTTGAAACAATTCAGACTCCCACTAGAAGAAACAGAACTTGAAAAAAGGATAATTGTGGATGACACCTCAACCCAGTGGTCCAAAAACATGAAACATTTGACCCCGAGCACCCTCACACAGATAGACTACAATGAGAAGGAGAAAGGGGCCATTACTCAGTCTCCCTTATCAGATTGCCTTACGAGGAGTCATAGCATCCCTCAAGCAAATAGATCTCCATTACCCATTGCAAAGGTATCATCATTTCCATCTATTAGACCTATATATCTGACCAGGGTCCTATTCCAAGACAACTCTTCTCATCTTCCAGCAGCATCTTATAGAAAGAAAGATTCTGGGGTCCAAGAAAGCAGTCATTTCTTACAAGGAGCCAAAAAAAATAACCTTTCTTTAGCCATTCTAACCTTGGAGATGACTGGTGATCAAAGAGAGGTTGGCTCCCTGGGGACAAGTGCCACAAATTCAGTCACATACAAGAAAGTTGAGAACACTGTTCTCCCGAAACCAGACTTGCCCAAAACATCTGGCAAAGTTGAATTGCTTCCAAAAGTTCACATTTATCAGAAGGACCTATTCCCTACGGAAACTAGCAATGGGTCTCCTGGCCATCTGGATCTCGTGGAAGGGAGCCTTCTTCAGGGAACAGAGGGAGCGATTAAGTGGAATGAAGCAAACAGACCTGGAAAAGTTCCCTTTCTGAGAGTAGCAACAGAAAGCTCTGCAAAGACTCCCTCCAAGCTATTGGATCCTCTTGCTTGGGATAACCACTATGGTACTCAGATACCAAAAGAAGAGTGGAAATCCCAAGAGAAGTCACCAGAAAAAACAGCTTTTAAGAAAAAGGATACCATTTTGTCCCTGAACGCTTGTGAAAGCAATCATGCAATAGCAGCAATAAATGAGGGACAAAATAAGCCCGAAATAGAAGTCACCTGGGCAAAGCAAGGTAGGACTGAAAGGCTGTGCTCTCAAAACCCACCAGTCTTGAAACGCCATCAACGGGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGACTATGATGATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACACGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGGATTATGGGATGAGTAGCTCCCCACATGTTCTAAGAAACAGGGCTCAGAGTGGCAGTGTCCCTCAGTTCAAGAAAGTTGTTTTCCAGGAATTTACTGATGGCTCCTTTACTCAGCCCTTATACCGTGGAGAACTAAATGAACATTTGGGACTCCTGGGGCCATATATAAGAGCAGAAGTTGAAGATAATATCATGGTAACTTTCAGAAATCAGGCCTCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGGAAGATCAGAGGCAAGGAGCAGAACCTAGAAAAAACTTTGTCAAGCCTAATGAAACCAAAACTTACTTTTGGAAAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGGGCTTATTTCTCTGATGTTGACCTGGAAAAAGATGTGCACTCAGGCCTGATTGGACCCCTTCTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGGAATTTGCTCTGTTTTTCACCATCTTTGATGAGACCAAAAGCTGGTACTTCACTGAAAATATGGAAAGAAACTGCAGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAATTATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGGCTTAGTAATGGCTCAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCTATTCATTTCAGTGGACATGTGTTCACTGTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTATCCAGGTGTTTTTGAGACAGTGGAAATGTTACCATCCAAAGCTGGAATTTGGCGGGTGGAATGCCTTATTGGCGAGCATCTACATGCTGGGATGAGCACACTTTTTCTGGTGTACAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTTCAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTGTTGGCACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGCCTCTACATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATAAAACACAATATTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCATTATAGCATTCGCAGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGCATGCCATTGGGAATGGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTACTTTACCAATATGTTTGCCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGGAGTAATGCCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGGAGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTTCAGAATGGCAAAGTAAAGGTTTTTCAGGGAAATCAAGACTCCTTCACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGACTCGCTACCTTCGAATTCACCCCCAGAGTTGGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTCTACTGATTAATTAATAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTGCAATGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACACTACACAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTACTTAAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACACCTGCAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCGCCGCTAGCAGCTGATGCATAGCATGCGGTACCGGGAGATGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACTACGGGTACGTAGGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGAACAAACGATGCTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCAAGCGCCGCGACGGCCGAGGTCTACCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGCACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAAATTGTCACAACGCCGCGAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGCACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACAGCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTCATAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACACCCGCTTACGCAGGGCATCCATTTATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCTGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCAAATAAGCGTTGATATTCAGTCAATTACAAACATTAATAACGAAGAGATGACAGAAAAATTTTCATTCTGTGACAGAGAA

ceDNA Vector Name Elements arranged from 5′ to 3′ (Left ITR to RightITR) ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site-hAAT_PromoterSet- vector 1PmeI_site-Mod_Minimum_Consensus_Kozak-hFactorVIII_WT_ORF_v2-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site-hAAT_PromoterSet- vector 2PmeI_site-Mod_Minimum_Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site-LPl_Promoter_Set- vector 3PmeI_site-Mod_Minimum_Consensus_Kozak-hFactorVIII_WT_ORF_v2-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site-LPl_Promoter_Set- vector 4PmeI_site-Mod_Minimum_Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site- vector 5hEF1a_FullLength_Promoter-PmeI_site-Mod_Minimum_Consensus_Kozak-hFactorVIII_WT_ORF_v2-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site- vector 6hEF1a_FullLength_Promoter-PmeI_site-Mod_Minimum_Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site-LPl_Promoter_Set- vector 7PmeI_site-Mod_Minimum_Consensus_Kozak-hFactorVIII_206variant_ORF_v1-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-NotI_site- vector 8hEF1a_FullLength_Promoter-PmeI_site-Mod_Minimum_Consensus_Kozak-hFactorVIII_206variant_ORF_v1-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v2.1-CMV_enhancer-gB-actin_Promoter- vector9 Consensus_Kozak-XbaLsite-PmeI_site-Mod_Minimum_Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-mir-142_3pUTR-bGH-spacer_right-ITR_v2.1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v2.1-hAAT_PromoterSet-PmeI_site-vector 10 Mod_Minimum_Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-mir-142_3pUTR-bGH-spacer_right-ITR_v2.1-right-ITR_v1ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v2.1-VandenDriessche_PromoterSet- vector 11PmeI_site-Mod_Minimum_Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-BMN270_Enhancer- vector 12BMN270_Promoter-BMN270_5pUTR-BMN270_hFVIII-BDD-SQ-BMN270_polyA-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-SPK8011_Promoter-SPK8011_5pUTR- vector 13SPK8011_hFVIII-BDD-SQ-SPK8011_polyA-spacer_right-ITR_v1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v1-Minimum_Consensus_Kozak-vector 14 TTR_liver_specific_Promoter-SangamoCRMSBS2-Intron3_5pUTR-SangamoCRMSBS2-Intron3_hFVIII-BDD-SQ-SangamoCRMSBS2_polyA-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-hAAT_PromoterSet-PmeI_site- vector 15Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-hAAT_PromoterSet-PmeI_site- vector 16Consensus_Kozak-Nathwani_hFVIII-BDD-V3-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-hAAT_PromoterSet-PmeI_site- vector 17Consensus_Kozak-Nathwani_hFVIII-BDD-V3-PacI_site-WPRE_3pUTR-mir-142_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v1-VandenDriessche_PromoterSet-vector 18 PmeI_site-Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-VandenDriessche_PromoterSet- vector 19PmeI_site-Consensus_Kozak-Nathwani_hFVIII-BDD-V3-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v1-VandenDriessche_PromoterSet- vector 20PmeI_site-Consensus_Kozak-Nathwani_hFVIII-BDD-V3-PacI_site-WPRE_3pUTR-mir-142_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v1-NotI_site-CAG_PromoterSet-vector 21 PmeI_site-Consensus_Kozak-hVIII-BDD-SQ_codopOZ-PacI_site-WPRE_3pUTR-mir-142_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v1-CET_PromoterSet-PmeI_site-vector 22 Consensus_Kozak-Nathwani_hFVIII-BDD-V3-PacI_site-WPRE_3pUTR-mir-142_3pUTR-bGH-XbaLsite-spacer_right- ITR_v1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v1-VandenDriessche_PromoterSet-vector 23 PmeI_site-Consensus_Kozak-hFVIII-226variant-F309S_CpGmin-codop_ORF-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1ceDNAFVIII- left-ITR_v1-spacer_left-ITR_v1-VandenDriessche_PromoterSet-vector 24 PmeI_site-Consensus_Kozak-Endo_hFVIII_cDNA-PacI_site-WPRE_3pUTR-bGH-spacer_right-ITR_v1-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v2-NdeLsite-CET_PromoterSet- vector 25PmeI_site-Mod_Minimum_Consensus_Kozak-hFactorVIII-BDD_CpGmin-codop_ORF-PacI_site-HBB_3pUTR-mir-142_3pUTR-SV40_polyA-spacer_right-ITR_v2-right-ITR_v1 ceDNAFVIII-left-ITR_v1-spacer_left-ITR_v2-NdeLsite-CET_PromoterSet- vector 26PmeI_site-Mod_Minimum_Consensus_Kozak-Endo_hFVIII_cDNA-PacI_site-HBB_3pUTR-mir-142_3pUTR-SV40_polyA-spacer_right-ITR_v2-right-ITR_v1

Test articles were supplied in a concentrated stock, and stored atnominally 4° C. Formulations were not vortexed or centrifuged. Groupswere housed in clear polycarbonate cages with contact bedding on aventilated rack in a procedure room. Food and filtered tap wateracidified with 1N HCl to a targeted pH of 2.5-3.0 were provided to theanimals ad libitum. Blood was collected at interim and terminal timepoints as set forth below:

Sample Collection Times Group Saphenous or Orbital Bleed Terminal NumberPlasma 1-10 Days 3 Day 7 Volume/ 120 μL whole blood MOV PortionProcessing/ 120 μI, whole blood was 600 μI, whole blood Storage added totube pre-coated was added to tube with 13.33 μL of 3.2% pre-coated with66.6 μI, of 3.2% sodium citrate and kept sodium citrate and kept ambientuntil processed ambient until processed One (1) aliquot of plasma Four(4) aliquots of plasma Frozen at nominally −70° C. Frozen at nominally−70° C.

Blood sample collection was as follows:

Volume (mL) Sample D28 Whole Blood Destination D −3 (~) D 3, 7, 14, 21(terminal) 0.15 cytokine 0.05 PHE assay × 2 0.05 0.05 0.6 total/day (mL)0.05 0.05 0.6

Study Details are Provided as Follows:

Species (number, sex, age): C57BL/6 (JAX 000664) mice (N=40 and 4 spare,male, 6 weeks of age at arrival) from Jackson Laboratories.

Class of Compound: ceDNA.

Cage Side Observations: Cage side observations were performed daily.

Clinical Observations: Clinical observations were performed 1, 5-6 and24 hours post dose on Day 0.

Body Weights: Body weight for all animals was recorded on Days 0, 3 and7, including prior to euthanasia.

Dose Formulation: Test articles were supplied in a concentration stock.Stock was diluted with PBS immediately prior to use. Prepared materialswere stored at ˜4° C. (or on wet ice) if dosing was not performedimmediately.

Dose Administration: Test Materials were dosed on Day 0 by hydrodynamicIV administration, at a set volume per animal, 90-100 ml/kg (dependenton the lightest animal in the group) via lateral tail vein (dosed within5 seconds).

Blood Collection: Animals had interim blood collected on Day 3 per thetable above. After each collection, animals received 0.5-1.0 mL lactatedRinger's, subcutaneously Animals had blood collected according to theblood collection tables above. For plasma collections, whole blood wascollected into non-coated Eppendorf style tubes. Immediately 120 μL waswithdrawn and placed into tubes containing 13.33 μL of 3.2% sodiumcitrate. Blood was gently mixed and maintained ambient until processed.Whole blood samples were centrifuged at 2,000 g for 15 minutes underambient conditions (20-25° C.). Plasma samples were withdrawn avoidingthe cell pack. One (1) aliquot was made and any clotting in the wholeblood sample or hemolysis in the plasma was noted. All samples werestored at nominally −70° C. All samples were stored at nominally −70° C.until shipped.

Anesthesia Recovery: Animals were monitored continuously while underanesthesia, during recovery and until mobile.

Euthanasia & Terminal Blood Collection: On Day 7, animals wereeuthanized by CO₂ asphyxiation followed by thoracotomy andexsanguination. Maximum obtainable blood volume was collected by cardiacpuncture and processed to serum per facility SOPS and stored in two (2)aliquots. For plasma collections, whole blood was collected by syringeand 600 μL, placed immediately in tubes containing 66.66 μL of 3.2%sodium citrate. Blood was gently mixed and maintained ambient untilprocessed. Whole blood samples were centrifuged at 2,000 g for 15minutes under ambient conditions (20-25° C.). Plasma samples werewithdrawn avoiding the cell pack and four (4) aliquots made. Anyclotting in the whole blood sample or hemolysis in the plasma was noted.

Results:

The results shown in FIGS. 12-15 show the effect of the various ceDNAvectors expressing FVIII on serum FVIII levels, where detection of FVIIIin the serum indicated that the ceDNA vector was able to express FVIIIafter injection. FIG. 12 is a graph that depicts plasma FVIIIconcentration (IU/ml) 3 days after hydrodynamic delivery of variousceDNA vectors expressing FVIII (ceDNAFVIII-vector 4, ceDNAFVIII-vector6, ceDNAFVIII-vector 12, ceDNAFVIII-vector 14, ceDNAFVIII-vector 16,ceDNAFVIII-vector 18, ceDNAFVIII-vector 19, ceDNAFVIII-vector 21,ceDNAFVIII-vector 22). ceDNAFVIII-vector 16, ceDNAFVIII-vector 19 andceDNAFVIII-vector 21 showed the highest plasma Factor VIII concentrationafter 3 days. Vehicle only was used as control. FIG. 13 is a graph thatdepicts plasma FVIII concentration (IU/ml) 3 days after hydrodynamicdelivery of various ceDNA vectors expressing FVIII (ceDNAFVIII-vector 1,ceDNAFVIII-vector 2, ceDNAFVIII-vector 3, ceDNAFVIII-vector 5,ceDNAFVIII-vector 7, ceDNAFVIII-vector 8, ceDNAFVIII-vector 12,ceDNAFVIII-vector 13, ceDNAFVIII-vector 15). ceDNAFVIII-vector 8 showedthe highest plasma Factor VIII concentration after 3 days. Vehicle onlywas used as control. FIG. 14 is a graph that depicts plasma FVIIIconcentration (IU/ml) 3 days after hydrodynamic delivery of variousceDNA vectors expressing FVIII (ceDNAFVIII-vector 9, ceDNAFVIII-vector10, ceDNAFVIII-vector 11, ceDNAFVIII-vector 12, ceDNAFVIII-vector 17,ceDNAFVIII-vector 20, ceDNAFVIII-vector 24, ceDNAFVIII-vector 25,ceDNAFVIII-vector 26). ceDNAFVIII-vector 17 and ceDNAFVIII-vector 20showed the highest plasma Factor VIII concentration after 3 days.Vehicle only was used as control. FIG. 15 is a graph that depicts plasmaFVIII concentration (IU/ml) 1 day after hydrodynamic delivery ofescalating doses of ceDNAFVIII-vector 23 (0.005-50 μg). As shown in FIG.15, plasma FVIII increased in a dose-dependent manner with increasingdosage of ceDNAFVIII-vector 23. Vehicle only was used as control.

Example 14: FVIII Expression after LNP:ceDNA Delivery in Combinationwith a Kinase Inhibitor in Male CD-1 Mice

LNP:ceDNA vectors were prepared as described in Example 6. The effect ofceDNA:lipid formulations were tested in male CD-1 mice. The vehicle fordosing and inhibitor preparation was 0.5% methylcellulose.

LNP:ceDNA test material (ceDNAFVIII-vector 23) was administered asfollows:

Animals Dose Dose Group per Level Volume Treatment Terminal No. GroupTreatment (mg/kg (mL/kg) Regimen, IV Time Point 1 5 PBS 2.0 5 Once onDay 0 Day 7 2 5 ceDNAFVIII-vector 23 2.0 Test article storage (stockformulations): Comments: Test article for Groups 1-2 was suppliedResidual TA, both diluted and stock, was in a concentrated stock (1.0mg/mL) and stored at nominally 4° C. until dosing is stored nominally−80° C. until use. complete, then discarded. Do not vortex or centrifugeformulations. Chow/Water: Mouse Diet 5058 and filtered Housing: Grouphoused in clear tap water acidified with 1N HCl to a targetedpolycarbonate cages with contact bedding pH of 2.5-3.0 were provided tothe animals in a ventilated rack in a procedure room. ad libitum. No. =Number; IV = intravenous; ROA = route of administration

Blood (interim and terminal) samples were collected as set forth below:

Sample Collection Times Group Whole Blood (orbital) Terminal Whole Blood(cardiac) Number Sodium Citrate Plasma 1-2 Day 3 Day 7 Volume/ 120 Awhole blood MOV Portion Processing/ whole blood was added to 600 μ 1_,whole blood was Storage tube pre-coated with 13.33 added to tubepre-coated with pi of 3.2% sodium citrate 66.6 1. μL of 3.2% sodium andkept ambient citrate and kept ambient until processed until processedOne (1) aliquot of plasma Two (2) aliquots of plasma Frozen at nominally−70° C. Frozen at nominally −70° C.

Tissue samples were collected as set forth below:

Group Sample Collection Times Number Liver 1-2 On Day 7 Volume/PortionWhole organ, divided Processing Whole organ weight 4 × = 25-50 mg piecesweighed and snap frozen individually (Lake Pharma) Storage Frozen atnominally −70° C.

Study Details are Provided as Follows:

Species (number, sex, age): CD-1 mice (N=10+2 spare, male, 4 weeks ofage at arrival) from Charles River Laboratories.

Cage Side Observations: Cage side observations were performed daily.

Clinical Observations: Clinical observations were performed about 1, 5-6and 24 hours post Day 0 Test Material dose.

Body Weights: Body weight for all animals was recorded on Days 0, 1, 2,3, and 7, including prior to euthanasia.

Test Material Dose Formulation: Test articles were supplied in aconcentration stock. Stock diluted with PBS immediately prior to use.Prepared materials were stored at −4° C. (or on wet ice) if dosing wasnot performed immediately.

Dose Administration: Test articles were dosed at 5 mL/kg on Day 0 forGroups 1-2 by intravenous administration via lateral tail vein.

Blood Collection: All animals in Groups 1-2 had interim blood collectedon Day 3 per blood collection table above. After each collection animalsreceived 0.5-1.0 mL lactated Ringer's, subcutaneously. For plasmacollections, whole blood was collected into non-coated Eppendorf styletubes via orbital sinus puncture under anesthesia per facility SOPSImmediately 120 μL was withdrawn and placed into tubes containing 13.33pl of 3.2% sodium citrate. Blood was gently mixed and maintained ambientuntil processed. Whole blood samples were centrifuged at 2,000 g for 15minutes under ambient conditions (20-25° C.). Plasma samples werewithdrawn avoiding the cell pack. One (1) aliquot was made and anyclotting in the whole blood sample or hemolysis in the plasma will benoted.

Anesthesia Recovery: Animals were monitored continuously while underanesthesia, during recovery and until mobile.

Terminal Blood Collection: On Day 7, animals were euthanized by CO₂asphyxiation followed by thoracotomy and exsanguination. Maximumobtainable blood volume was collected by cardiac puncture and processedto plasma. For plasma collections, whole blood was collected by syringeand 600 μL placed immediately tubes containing 66.66 μL, of 3.2% sodiumcitrate. Blood was gently mixed and maintained ambient until processed.Whole blood samples were centrifuged at 2,000 g for 15 minutes underambient conditions (20-25° C.). Plasma samples were withdrawn avoidingthe cell pack and two (2) aliquots made. Any clotting in the whole bloodsample or hemolysis in the plasma was noted.

Perfusion: Following exsanguination, all animals underwent cardiacperfusion with saline. In brief, whole body intracardiac perfusion wasperformed by inserting 23/21-gauge needle attached to 10 mL syringecontaining saline into the lumen of the left ventricle for perfusion.The right atrium was incised to provide a drainage outlet for perfusate.Gentle and steady pressure was applied to the plunger to perfuse theanimal after the needle has been positioned in the heart. Adequate flowof the flushing solution was ensured until the exiting perfusate flowsclear (free of visible blood) indicating that the flushing solution hadsaturated the body and the procedure was complete.

Tissue Collection: Terminal tissues were collected from moribund animalsthat were euthanized prior to their scheduled time point. If possible,tissues were collected and stored from animals that were found dead.

After euthanasia and perfusion, the liver was harvested. From the liver,four (4) 25-50 mg sections were collected and weighed. Sections weresnap frozen individually, stored at nominally −70° C. until shipped.

Results: FIG. 16 shows plasma FVIII concentration (IU/mL) at 7 daysafter administration of the LNP:ceDNAFVIII-vector 23 test article. Asshown in FIG. 16, FVIII was readily detected in plasma samples from micetreated with the LNP:ceDNAFVIII-vector 23 test article, but was notobserved in mice treated with vehicle only.

REFERENCES

All publications and references, including but not limited to patentsand patent applications, cited in this specification and Examples hereinare incorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in the manner described above forpublications and references.

1. A capsid-free close-ended DNA (ceDNA) vector comprising: at least oneheterologous nucleotide sequence between flanking inverted terminalrepeats (ITRs), wherein at least one heterologous nucleotide sequenceencodes at least one FVIII protein, wherein the least one heterologousnucleotide sequence that encodes at least one FVIII protein is selectedfrom a sequence having at least 85% identity to any sequence in Table 1.2. The ceDNA vector of claim 1, wherein the ceDNA vector comprises anucleic acid sequence selected from a sequence having at least 85%identity with a sequence in Table
 9. 3. The ceDNA vector of claim 1 orclaim 2, wherein the ceDNA vector comprises a nucleic acid sequence thatis at least 85% identical to SEQ ID NO:
 210. 4. The ceDNA vector ofclaim 1 or claim 2, wherein the ceDNA vector comprises a nucleic acidsequence that is at least 85% identical to SEQ ID NO:
 214. 5. The ceDNAvector of any of claims 1-4, wherein the ceDNA vector comprises apromoter operatively linked to the least one heterologous nucleotidesequence that encodes at least one FVIII protein.
 6. The ceDNA vector ofany of claims 1-5, wherein the ceDNA vector comprises an enhancer. 7.The ceDNA vector of any of claims 1-6, wherein the ceDNA vectorcomprises a 5′ UTR and/or intron sequence.
 8. The ceDNA vector of any ofclaims 1-7, wherein the ceDNA vector comprises a 3′ UTR sequence.
 9. TheceDNA vector of any of claims 1-8, wherein the ceDNA vector comprises atleast one poly A sequence.
 10. The ceDNA vector of any one of claims1-9, wherein the ceDNA vector comprises at least one promoter operablylinked to at least one heterologous nucleotide sequence.
 11. The ceDNAvector of any one of claims 1-10, wherein at least one heterologousnucleotide sequence is cDNA.
 12. The ceDNA vector of any one of claims1-11, wherein at least one ITR comprises a functional terminalresolution site and a Rep binding site.
 13. The ceDNA vector of any oneof claims 1-12, wherein one or both of the ITRs are from a virusselected from a parvovirus, a dependovirus, and an adeno-associatedvirus (AAV).
 14. The ceDNA vector of any one of claims 1-13, wherein theflanking ITRs are symmetric or asymmetric.
 15. The ceDNA vector of claim14, wherein the flanking ITRs are symmetrical or substantiallysymmetrical.
 16. The ceDNA vector of claim 14, wherein the flanking ITRsare asymmetric.
 17. The ceDNA vector of any one of claims 1-16, whereinone or both of the ITRs are wild type, or wherein both of the ITRs arewild-type.
 18. The ceDNA vector of any one of claims 1-17, wherein theflanking ITRs are from different viral serotypes.
 19. The ceDNA vectorof any one of claims 1-18, wherein the flanking ITRs are from a pair ofviral serotypes shown in Table
 2. 20. The ceDNA vector of any one ofclaims 1-19, wherein one or both of the ITRs comprises a sequenceselected from the sequences in Table 3, Table 5A, Table 5B, or Table 6.21. The ceDNA vector of any one of claims 1-20, wherein at least one ofthe ITRs is altered from a wild-type AAV ITR sequence by a deletion,addition, or substitution that affects the overall three-dimensionalconformation of the ITR.
 22. The ceDNA vector of any one of claims 1-21,wherein one or both of the ITRs are derived from an AAV serotypeselected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, and AAV12.
 23. The ceDNA vector of any one of claims 1-22,wherein one or both of the ITRs are synthetic.
 24. The ceDNA vector ofany one of claims 1-23, wherein one or both of the ITRs is not a wildtype ITR, or wherein both of the ITRs are not wild-type.
 25. The ceDNAvector of any one of claims 1-24, wherein one or both of the ITRs ismodified by a deletion, insertion, and/or substitution in at least oneof the ITR regions selected from A, A′, B, B′, C, C′, D, and D′.
 26. TheceDNA vector of claim 25, wherein the deletion, insertion, and/orsubstitution results in the deletion of all or part of a stem-loopstructure normally formed by the A, A′, B, B′ C, or C′ regions.
 27. TheceDNA vector of any one of claims 1-26, wherein one or both of the ITRsare modified by a deletion, insertion, and/or substitution that resultsin the deletion of all or part of a stem-loop structure normally formedby the B and B′ regions.
 28. The ceDNA vector of any one of claims 1-26,wherein one or both of the ITRs are modified by a deletion, insertion,and/or substitution that results in the deletion of all or part of astem-loop structure normally formed by the C and C′ regions.
 29. TheceDNA vector of any one of claims 1-26, wherein one or both of the ITRsare modified by a deletion, insertion, and/or substitution that resultsin the deletion of part of a stem-loop structure normally formed by theB and B′ regions and/or part of a stem-loop structure normally formed bythe C and C′ regions.
 30. The ceDNA vector of any one of claims 1-29,wherein one or both of the ITRs comprise a single stem-loop structure inthe region that normally comprises a first stem-loop structure formed bythe B and B′ regions and a second stem-loop structure formed by the Cand C′ regions.
 31. The ceDNA vector of any one of claims 1-30, whereinone or both of the ITRs comprise a single stem and two loops in theregion that normally comprises a first stem-loop structure formed by theB and B′ regions and a second stem-loop structure formed by the C and C′regions.
 32. The ceDNA vector of any one of claims 1-31, wherein one orboth of the ITRs comprise a single stem and a single loop in the regionthat normally comprises a first stem-loop structure formed by the B andB′ regions and a second stem-loop structure formed by the C and C′regions.
 33. The ceDNA vector of any one of claims 1-32, wherein bothITRs are altered in a manner that results in an overallthree-dimensional symmetry when the ITRs are inverted relative to eachother.
 34. The ceDNA vector of any one of claims 1-33, wherein one orboth of the ITRs comprises a sequence selected from the sequences inTables 3, 5A, 5B, and
 6. 35. The ceDNA vector of any one of claims 1-34,wherein at least one heterologous nucleotide sequence is under thecontrol of at least one regulatory switch.
 36. The ceDNA vector of claim35, wherein at least one regulatory switch is selected from a binaryregulatory switch, a small molecule regulatory switch, a passcoderegulatory switch, a nucleic acid-based regulatory switch, apost-transcriptional regulatory switch, a radiation-controlled orultrasound controlled regulatory switch, a hypoxia-mediated regulatoryswitch, an inflammatory response regulatory switch, a shear-activatedregulatory switch, and a kill switch.
 37. A method of expressing anFVIII protein in a cell comprising contacting the cell with the ceDNAvector of any one of claims 1-36.
 38. The method of claim 37, whereinthe cell is a photoreceptor or a RPE cell.
 39. The method of claim 37 or38, wherein the cell in in vitro or in vivo.
 40. The method of any oneof claims 37-39, wherein the at least one heterologous nucleotidesequence codon optimized for expression in the eukaryotic cell.
 41. Themethod of any one of claims 37-40, wherein the at least one heterologousnucleotide sequence is a sequence having at least 85% identity to anysequence set forth in Table
 1. 42. A method of treating a subject withhemophilia A, comprising administering to the subject a ceDNA vector ofany one of claims 1-36, wherein at least one heterologous nucleotidesequence encodes at least one FVIII protein.
 43. The method of claim 42,wherein levels of FVIII in the serum of the subject are increased insubjects administered the ceDNA vector compared to a control.
 44. Themethod of claim 43, wherein the increase in levels of FVIII is greaterthan about 40% compared to the control.
 45. The method of claim 42,wherein the at least one heterologous nucleotide sequence is a sequencehaving at least 85% identity to any sequence set forth in Table
 1. 46.The method of any of claims 42-45, wherein the ceDNA vector isadministered to a photoreceptor cell, or an RPE cell, or both.
 47. Themethod of any of claims 42-46, wherein the ceDNA vector expresses theFVIII protein in a photoreceptor cell, or an RPE cell, or both.
 48. Themethod of any of claims 42-47, wherein the ceDNA vector is administeredby any one or more of: subretinal injection, suprachoroidal injection orintravitreal injection.
 49. A pharmaceutical composition comprising theceDNA vector of any one of claims 1-36.
 50. A cell containing a ceDNAvector of any of claims 1-36.
 51. The cell of claim 50, wherein the cella photoreceptor cell, or an RPE cell, or both.
 52. A compositioncomprising a ceDNA vector of any of claims 1-36 and a lipid.
 53. Thecomposition of claim 52, wherein the lipid is a lipid nanoparticle(LNP).
 54. A kit comprising the ceDNA vector of any one of claims 1-36,the pharmaceutical composition of claim 49, the cell of claim 50 orclaim 51, or the composition of claim 52 or claim 53.